Anti-interferon-alpha antibodies

ABSTRACT

The present invention relates generally to the generation and characterization of neutralizing anti-IFN-α monoclonal antibodies with broad reactivity against various IFN-α subtypes. The invention further relates to the use of such anti-IFN-α antibodies in the diagnosis and treatment of disorders associated with increased expression of IFN-α, in particular, autoimmune disorders such as insulin-dependent diabetes mellitus (IDDM) and systemic lupus erythematosus (SLE).

This application claims the benefit under Title 35, United States Codes§119(e) of the U.S. provisional Application No. 60/270,775, filed onFeb. 22, 2001.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the generation andcharacterization of neutralizing anti-IFN-α monoclonal antibodies withbroad reactivity against various IFN-α subtypes. The invention furtherrelates to the use of such anti-IFN-α antibodies in the diagnosis andtreatment of disorders associated with increased expression of IFN-α, inparticular, autoimmune disorders such as insulin-dependent diabetesmellitus (IDDM) and systemic lupus erythematosus (SLE).

2. Description of the Related Art

Interferon-α (IFN-α)

Although interferons were initially discovered for their anti-viralactivities, subsequent research has unraveled a plethora of regulatoryactivities associated with these powerful cytokines. Type I interferonsform an ancient family of cytokines that includes IFN-α, IFN-β, IFN-δ,IFN-ω and IFN-τ (Roberts et al., J. Interferon Cytokine Res. 18: 805-816[1998]). They are coded by intronless genes and are widely distributedamongst vertebrates. Whereas IFN-β is coded by a single gene in primatesand rodents, more than 10 and 15 different subtypes of IFN-α have beenfound in mice and man respectively. Other interferons of type I are morerestricted, e.g. IFN-δ in the pig, IFN-τ in cattle and sheep, and IFN-ωin cattle and humans. Thus, human type I interferons comprise multiplemembers of the IFN-α family, and single members of the IFN-β and IFN-ωfamilies. All type I IFNs appear to bind to a single receptor that iscomprised of at least two membrane spanning proteins. Type IIinterferons on the other hand are represented by a single member, IFN-γ,and bind to a distinct receptor.

Although all type I IFNs, including IFN-α, exhibit anti-viral andanti-proliferative activities and thereby help to control viralinfections and tumors (Lefevre et al., Biochimie 80: 779-788 [1998];Horton et al., Cancer Res. 59: 4064-4068 [1999]; Alexenko et al., J.Interferon Cytokine Res. 17: 769-779 [1997]; Gresser, J. Leukoc. Biol.61: 567-574 [1997]), there are also several autoimmune diseases that areassociated with increased expression of IFNα, most notablyinsulin-dependent diabetes mellitus (IDDM) and systemic lupuserythematosus (SLE).

Type I diabetes, also known as autoimmune diabetes or insulin-dependentdiabetes mellitus (IDDM), is an autoimmune disease characterized by theselective destruction of pancreatic β cells by autoreactive Tlymphocytes (Bach, Endocr. Rev. 15: 516-542 [1994]; Castano andEisenbarth, Annu. Rev. Immunol. 8: 647-679 [1990]; Shehadeh andLafferty, Diabetes Rev. 1: 141-151 [1993]). The pathology of IDDM isvery complex involving an interaction between an epigenetic event(possibly a viral infection), the pancreatic β cells and the immunesystem in a genetically susceptible host. A number of cytokines,including IFN-α and IFN-γ, have been implicated in the pathogenesis ofIDDM in humans and in animal models of the disease (Campbell et al., J.Clin. Invest. 87: 739-742 [1991]; Huang et al., Diabetes 44: 658-664[1995]; Rhodes and Taylor, Diabetologia 27: 601-603 [1984]). Forexample, pancreatic Ifn-α mRNA expression and the presence ofimmunoreactive IFN-α in β cells of patients with IDDM have been reported(Foulis et al., Lancet 2: 1423-1427 [1987]; Huang et al., [1995] supra;Somoza et al., J. Immunol. 153: 1360-1377 [1994]). IFN-α expression hasbeen associated with hyperexpression of major histocompatibility complex(MHC) class I_(A) antigens in human islets (Foulis et al., [1987] supra;Somoza et al., [1994] supra). In two rodent models of autoimmunediabetes, the diabetes-prone DP-BB rat and streptozotocin-treated mice,Ifn-α mRNA expression in islets precedes insulitis and diabetes (Huanget al., Immunity 1: 469-478 [1994]). Furthermore, transgenic miceharboring a hybrid human insulin promoter-Ifn-α construct develophypoinsulinemic diabetes accompanied by insulitis (Stewart et al.,Science 260: 1942-1946 [1993]).

It appears that local expression of IFN-α by pancreatic islet cells inresponse to potential diabetogenic stimuli such as viruses may triggerthe insulitic process. Consistent with its role as an initiating agent,IFN-α has been shown to induce intercellular adhesion molecule-1(ICAM-1) and HLA class I_(A) on endothelial cells from human islets,which may contribute to leukocyte infiltration during insulitis(Chakrabarti et al., J. Immunol. 157: 522-528 [1996]). Furthermore,IFN-α facilitates T cell stimulation by the induction of theco-stimulatory molecules ICAM-1 and B7.2 on antigen-presenting cells inislets (Chakrabarti et al., Diabetes 45: 1336-1343 [1996]). Thesestudies collectively indicate that early IFN-α expression by β cells maybe a critical event in the initiation of autoimmune diabetes. Althoughthere are a number of reports implicating IFN-γ in the development ofIDDM in rodent models, there is a poor correlation between theexpression of this cytokine and human IDDM. Thus, cells expressing IFN-γcan be found in the islets of a subset of human patients selected forsignificant lymphocytic infiltration into the islets. In a group ofpatients that were not selected by this criterion there was no obviousassociation between IFN-γ expression and human IDDM.

Based on the increased level of IFN-α expression in patients withsystemic lupus erythematosus (SLE), IFN-α has also been implicated inthe pathogenesis of SLE (Ytterberg and Schnitzer, Arthritis Rheum. 25:401-406 [1982]; Shi et al., Br. J. Dermatol. 117: 155-159 [1987]). It isinteresting to note that IFN-α is currently used for the treatment ofcancer as well as viral infection such as chronic hepatitis due tohepatitis B or hepatitis C virus infection. Consistent with theobservations of increased levels of IFN-α triggering autoimmunity,significant increase in the appearance of autoimmune disorders such asIDDM, SLE and autoimmune thyroiditis has been reported in the patientsundergoing IFN-α therapy. For example, prolonged use of IFN-α as ananti-viral therapy has been shown to induce IDDM (Waguri et al.,Diabetes Res. Clin. Pract. 23: 33-36 [1994]; Fabris et al., J. Hepatol.28: 514-517 [1998]) or SLE (Garcia-Porrua et al., Clin. Exp. Rheumatol.16: 107-108 [1998]). The treatment of coxsackievirus B (CBV) infectionwith IFN-α therapy is also associated with the induction of IDDM(Chehadeh et al., J. Infect. Dis. 181: 1929-1939 [2000]). Similarly,there are multiple case reports documenting IDDM or SLE in IFN-α treatedcancer patients (Ronnblom et al., J. Intern. Med. 227: 207-210 [1990]).

Antibody Therapy

The use of monoclonal antibodies as therapeutics has gained increasedacceptance with several monoclonal antibodies (mAbs) either approved forhuman use or in late stage clinical trials. The first mAb approved bythe US Food and Drug Administration (FDA) for the treatment of allograftrejection was anti-CD3 (OKT3) in 1986. Since then the pace of progressin the field of mAbs has been considerably accelerated, particularlyfrom 1994 onwards which led to approval of additional seven mAbs forhuman treatment. These include ReoPro® for the management ofcomplications of coronary angioplasty in 1994, Zenapax® (anti-CD25) forthe prevention of allograft rejection in 1997, Rituxan® (anti-CD20) forthe treatment of B cell non-Hodgkin's lymphoma in 1997, Infliximab®(anti-TNF-α) initially for the treatment of Crohn's disease in 1998 andsubsequently for the treatment of rheumatoid arthritis in 1999,Simulect® (anti-CD25) for the prevention of allograft rejection in 1998,Synagis® (anti-F protein of respiratory syncitial virus) for thetreatment of respiratory infections in 1998, and Herceptin®(anti-HER2/neu) for the treatment of HER2 overexpressing metastaticbreast tumors in 1998 (Glennie and Johnson, Immunol. Today 21: 403-410[2000]).

Anti-IFN-α Antibodies

Disease states that are amenable to intervention with mAbs include allthose in which there is a pathological level of a target antigen. Forexample, an antibody that neutralizes IFN-α present in the sera ofpatients with SLE, and expressed by the pancreatic islets in IDDM, is apotential candidate for therapeutic intervention in these diseases. Itcould also be used for therapeutic intervention in other autoimmunediseases with underlying increase in and causative role of IFN-αexpression. In both human IDDM (Foulis, et al., Lancet 2: 1423-1427[1987]; Huang, et al., Diabetes 44: 658-664 [1995]; Somoza, et al., J.Immunol. 153: 1360-1377 [1994]) and human SLE (Hooks, et al., Arthritis& Rheumatism 25: 396-400 [1982]; Kim, et al., Clin. Exp. Immunol. 70:562-569 [1987]; Lacki, et al., J. Med. 28: 99-107 [1997]; Robak, et al.,Archivum Immunologiae et Therapiae Experimentalis 46: 375-380 [1998];Shiozawa, et al., Arthritis & Rheumatism 35: 417-422 [1992]; von Wussow,et al., Rheumatology International 8: 225-230 [1988]) there appears tobe correlation between disease and IFN-α but not with either IFN-β orIFN-γ. Thus, anti-interferon mAb intervention in IDDM or SLE wouldrequire specific neutralization of most, if not all, of the IFN-αsubtypes, without any significant neutralization of IFN-β or IFN-γ.Leaving the activity of these last two interferons intact may also havean advantage in allowing the retention of significant anti-viralactivity.

While a few mAbs that show reactivity with a range of recombinant humanIFN-α subtypes have been described, these were found to neutralize onlya limited subset of the recombinant IFN-α subtypes analyzed or were notcapable of neutralizing the mixture of IFN-α subtypes that are producedby stimulated peripheral blood leukocytes (Tsukui et al., Microbiol.Immunol. 30: 1129-1139 [1986]; Berg, J. Interferon Res. 4: 481-491[1984]; Meager and Berg, J. Interferon Res. 6: 729-736 [1986]; U.S. Pat.No. 4,902,618; and EP publication No. 0,139,676 B1).

Accordingly, there is a great need for anti-IFN-α antibodies that notonly bind to most, preferably all, subtypes of IFN-α but also neutralizesuch subtypes while do not interfere with the biological function ofother interferons.

SUMMARY OF THE INVENTION

The present invention is based on the development of a monoclonalantibody that was experimentally found to neutralize all seven ofdifferent recombinant human IFN-α subtypes tested and two independentpools of natural human IFN-α subtypes.

In one aspect, the invention provides an anti-human IFN-α monoclonalantibody which binds to and neutralizes a biological activity of atleast human IFN-α subtypes IFN-α1, IFN-═2, IFN-α4, IFN-α5, IFN-α8,IFN-α10, and IFN-α21. In a further aspect, the invention provides ananti-human IFN-α monoclonal antibody which binds to and neutralizes abiological activity of all human IFN-α subtypes. The antibody of theinvention can significantly reduce or eliminate a biological activity ofthe human IFN-α in question. In one embodiment, the antibody of theinvention is capable of neutralizing at least 60%, or at least 70%,preferably at least 75%, more preferably at least 80%, even morepreferably at least 85%, still more preferably at least 90%, still morepreferably at least 95%, most preferably at least 99% of a biologicalactivity of the subject human IFN-α. In another embodiment, the humanIFN-α biological activity-neutralizing monoclonal antibody does notneutralize the corresponding biological activity of human IFN-β.

The biological activity of the subject human IFN-α's may beIFNAR2-binding activity. In a particular embodiment, the inventionprovides an anti-human IFN-α monoclonal antibody is capable of bindingto and blocking at least 60%, or at least 70%, preferably at least 75%,more preferably at least 80%, even more preferably at least 85%, stillmore preferably at least 90%, still more preferably at least 95%, mostpreferably at least 99% of the IFNAR2-binding activity of all, orsubstantially all human IFN-α subtypes. In another embodiment, theinvention provides an anti-human IFN-α monoclonal antibody that iscapable of binding to and blocking at least 60%, or at least 70%,preferably at least 75%, more preferably at least 80%, even morepreferably at least 85%, still more preferably at least 90%, still morepreferably at least 95%, most preferably at least 99% of theIFNAR2-binding activity of each of human IFN-α subtypes 1, 2, 4, 5, 8,10 and 21. In another embodiment, the anti-human IFN-α monoclonalantibody does not cross-react with human IFN-β.

The biological activity of the subject human IFN-α's may be an antiviralactivity. In one embodiment, the anti-human IFN-α monoclonal antibody iscapable of binding to and neutralizing the antiviral activity of all, orsubstantially all human IFN-α subtypes. In another embodiment, theanti-human IFN-α monoclonal antibody is capable of binding to andneutralizing the antiviral activity of each of human IFN-α subtypes 1,2, 4, 5, 8, 10 and 21. In a particular embodiment, the inventionprovides an anti-human IFN-α monoclonal antibody that is capable ofbinding to and neutralizing at least 60%, or at least 70%, preferably atleast 75%, more preferably at least 80%, even more preferably at least85%, still more preferably at least 90%, still more preferably at least95%, most preferably at least 99% of the antiviral activity of all, orsubstantially all human IFN-α subtypes. In yet another embodiment, theinvention provides an anti-human IFN-α monoclonal antibody which bindsto and neutralizes at least 60%, or at least 70%, or at least 75%, or atleast 80%, or at least 85%, or at least 90%, or at least 95%, or atleast 99% of the antiviral activity of each of human IFN-α subtypes 1,2, 4, 5, 8, 10 and 21. In still another embodiment, the human IFN-αantiviral activity-neutralizing monoclonal antibody does not neutralizethe antiviral activity of human IFN-β.

The antibody may be a murine, humanized or human antibody. The antibodymay be the murine anti-human IFN-α monoclonal antibody 9F3 or ahumanized version of it such as version 13 (V13) or chimeric formthereof. The scope of the invention also covers an antibody that bindsessentially the same IFN-α epitope as murine anti-human IFN-α monoclonalantibody 9F3 or a humanized or chimeric form thereof. For example, areference antibody for this purpose is an anti-IFN-α antibody producedby the murine hybridoma cell line 9F3.18.5 deposited with ATCC on Jan.18, 2001 and having accession No. PTA-2917. In another embodiment, theinvention provides a murine or murine/human chimeric anti-human IFN-αmonoclonal antibody comprising the murine light chain variable domainamino acid sequence shown in FIG. 5A (SEQ ID NO:1) and/or the murineheavy chain variable domain amino acid sequence shown in FIG. 5B (SEQ IDNO:2). In yet another embodiment, the invention provides a humanizedanti-human IFN-α monoclonal antibody comprising the humanized lightchain variable domain amino acid sequence shown in FIG. 5A (SEQ ID NO:3)and/or the humanized heavy chain variable domain amino acid sequenceshown in FIG. 5B (SEQ ID NO:5).

Additionally provided is an anti-human IFN-α monoclonal antibody thatbinds essentially the same epitopes on human IFN-α subtypes 1, 2, 4, 5,8, 10 and 21 that are bound by murine anti-human IFN-α monoclonalantibody 9F3 or a humanized or chimeric form thereof. Further providedherein is an anti-human IFN-α monoclonal antibody that competes withmurine anti-human IFN-α monoclonal antibody 9F3 for binding to each ofhuman IFN-α subtypes 1, 2, 4, 5, 8, 10 and 21.

Also provided is an isolated nucleic acid molecule encoding any of theantibodies described herein, a vector comprising the isolated nucleicacid molecule, a host cell transformed with the nucleic acid molecule,and a method of producing the antibody comprising culturing the hostcell under conditions wherein the nucleic acid molecule is expressed toproduce the antibody and optionally recovering the antibody from thehost cell. The antibody may be of the IgG class and isotypes such asIgG₁, IgG₂, IgG₃, or IgG₄. The scope of the invention also coversantibody fragments such as Fv, scFv, Fab, F(ab′)₂, and Fab′ fragments.

In another aspect, the present invention provides an anti-human IFN-αmonoclonal antibody light chain or a fragment thereof, comprising thefollowing CDR's (as defined by Kabat, et al., Sequences of Proteins ofImmunological Interest, Fifth Edition, NIH Publication 91-3242, BethesdaMd. [1991], vols. 1-3): (a) L1 of the formula RASQSVSTSSYSYMH (SEQ IDNO: 7); (b) L2 of the formula YASNLES (SEQ ID NO: 8); and (c) L3 of theformula QHSWGIPRTF (SEQ ID NO: 9). The scope of the invention alsocovers the light chain variable domain of such anti-human IFN-αmonoclonal antibody light chain fragment. The scope of the inventionfurther includes an anti-human IFN-α monoclonal antibody light chainpolypeptide comprising the mouse/human chimeric light chain variabledomain amino acid sequence, or the entire chimeric light chainpolypeptide amino acid sequence, encoded by the XAIFN-ChLpDR1 vectordeposited with the ATCC on Jan. 9, 2001 and having accession No.PTA-2880. The scope of the invention additionally includes an anti-humanIFN-α monoclonal antibody light chain polypeptide comprising thehumanized light chain variable domain amino acid sequence, or the entirehumanized light chain polypeptide amino acid sequence, encoded by theVLV30-IgG vector deposited with the ATCC on Jan. 9, 2001 and havingaccession No. PTA-2882.

In yet another aspect, the invention provides an anti-human IFN-αmonoclonal antibody heavy chain or a fragment thereof, comprising thefollowing CDR's: (a) H1 of the formula GYTFT EYIIH (SEQ ID NO: 10); (b)H2 of the formula SINPDYDITNYNQRFKG (SEQ ID NO: 11); and (c) H3 of theformula WISDFFDY (SEQ ID NO: 12). The scope of the invention also coversthe heavy chain variable domain of such anti-human IFN-α monoclonalantibody heavy chain fragment. The scope of the invention furtherincludes an anti-human IFN-α monoclonal antibody heavy chain polypeptidecomprising the mouse/human chimeric heavy chain variable domain aminoacid sequence, or the entire chimeric heavy chain polypeptide amino acidsequence, encoded by the XAIFN-ChHpDR2 vector deposited with the ATCC onJan. 9, 2001 and having accession No. PTA-2883. Additionally included isan anti-human IFN-α monoclonal antibody heavy chain polypeptidecomprising the humanized heavy chain variable domain amino acidsequence, or the entire humanized heavy chain polypeptide amino acidsequence, encoded by the vector VHV30-IgG2 deposited with the ATCC onJan. 9, 2001 and having accession No. PTA-2881.

In a further aspect, the invention provides an anti-human IFN-αmonoclonal antibody comprising (A) at least one light chain or afragment thereof, comprising the following CDR's: (a) L1 of the formulaRASQSVSTSSYSYMH (SEQ ID NO: 7); (b) L2 of the formula YASNLES (SEQ IDNO: 8); and (c) L3 of the formula QHSWGIPRTF (SEQ ID NO: 9); and (B) atleast one heavy chain or a fragment thereof, comprising the followingCDR's: (a) H1 of the formula GYTFTEYIIH (SEQ ID NO: 10); (b) H2 of theformula SINPDYDITNYNQRFKG (SEQ ID NO: 11); and (c) H3 of the formulaWISDFFDY (SEQ ID NO: 12). The antibody may be a homo-tetramericstructure composed of two disulfide-bonded antibody heavy chain-lightchain pairs. The scope of the invention specifically includes a linearantibody, a murine antibody, a chimeric antibody, a humanized antibody,or a human antibody. Further provided is a chimeric antibody comprising(1) the mouse/human chimeric light chain variable domain amino acidsequence, or the entire chimeric light chain polypeptide amino acidsequence, encoded by the XAIFN-ChLpDR1 vector deposited with the ATCC onJan. 9, 2001 and having accession No. PTA-2880; and (2) the mouse/humanchimeric heavy chain variable domain amino acid sequence, or the entirechimeric heavy chain polypeptide amino acid sequence, encoded by theXAIFN-ChHpDR2 vector deposited with the ATCC on Jan. 9, 2001 and havingaccession No. PTA-2883. Additionally provided herein is a humanizedantibody comprising (1) the humanized light chain variable domain aminoacid sequence, or the entire humanized light chain polypeptide aminoacid sequence, encoded by the VLV30-IgG vector deposited with the ATCCon Jan. 9, 2001 and having accession No. PTA-2882; and (2) the humanizedheavy chain variable domain amino acid sequence, or the entire humanizedheavy chain polypeptide amino acid sequence, encoded by the vectorVHV30-IgG2 deposited with the ATCC on Jan. 9, 2001 and having accessionNo. PTA-2881.

In yet another aspect, the invention provides a pharmaceuticalcomposition comprising an effective amount of the antibody of theinvention in admixture with a pharmaceutically acceptable carrier.

In a different aspect, the invention provides a method for diagnosing acondition associated with the expression of IFN-α, in a cell, comprisingcontacting the cell with an anti-IFN-α antibody, and detecting thepresence of IFN-α.

In yet another aspect, the invention provides a method for the treatmentof a disease or condition associated with the expression of IFN-α in apatient, comprising administering to the patient an effective amount ofan anti-IFN-α antibody. The patient is a mammalian patient, preferably ahuman patient. The disease is an autoimmune disease, such asinsulin-dependent diabetes mellitus (IDDM); systemic lupus erythematosus(SLE); or autoimmune thyroiditis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of the strategy used for thedevelopment of the anti-human IFN-α monoclonal antibodies.

FIG. 2 shows that a murine anti-human IFN-α mAb (9F3) is able toneutralize a spectrum of recombinant IFN-α subtypes but not recombinantIFN-β. The indicated IFN's were assayed for inhibition ofencephalomyocarditis (EMC) viral growth in A549 cells in the presence ofincreasing concentrations of the mAb 9F3. Data are presented as thepercentage of the viral growth inhibition activity obtained with theindicated IFN in the absence of mAb 9F3.

FIGS. 3A-3B show the neutralization of leukocyte interferon (Sigma)(FIG. 3A) and lymphoblastoid interferon (NIH reference Ga23-901-532)(FIG. 3B). In FIG. 3A, 20,000 IU/ml (filled bars) or 5,000 IU/ml (openbars) of leukocyte interferon (Sigma Product No. 1-2396) were incubatedwith blank control (buffer only) (denoted as “-”), 10 μg/ml controlmouse IgG (denoted as “mIgG”), or 10 μg/ml mAb 9F3 (denoted as “9F3”).Dilutions were assayed and the amount of remaining activity shown. Theresults shown are means of duplicate determinations. In FIG. 3B,lymphoblastoid interferon (NIH reference Ga23-901-532) was assayed at 10(filled columns) or 3 (open columns) IU/ml in the presence or absence ofthe indicated concentrations of mAb 9F3. A higher cytopathic effect isindicative of a decrease in interferon activity. The results shown arethe means of duplicate determinations.

FIG. 4 depicts results of an electrophoretic mobility shift assay (EMSA)showing the induction of an ISGF3/ISRE complex by IFN-α and the abilityof 9F3 mAb to prevent the formation of the complex. EMSA was performedin the presence or absence of either human IFN-α2 (denoted as “α2”) orIFN-β (denoted as “β”) at a concentration of 25 ng/ml with 9F3 mAb(denoted as “9F3”) or murine IgG control antibody (denoted as “IgG”) ata concentration of 10 μg/ml.

FIG. 5A shows the alignment of light chain variable domain amino acidsequences of murine 9F3 (murine, SEQ ID NO: 1), humanized 9F3 version 13(V13, SEQ ID NO: 3), and the consensus human variable domain light κsubgroup I (huκI, SEQ ID NO: 4). The CDRs (L1, SEQ ID NO: 7; L2, SEQ IDNO: 8; and L3, SEQ ID NO: 9) are highlighted by underlining. The residuenumbering is according to Kabat et al., (1991) supra. The differencesbetween the murine 9F3 and V13 sequences and the differences between 9F3and huκI sequences are indicated by asterisks.

FIG. 5B shows the alignment of heavy chain variable domain amino acidsequences of murine 9F3 (murine, SEQ ID NO: 2), humanized 9F3 version 13(V13, SEQ ID NO: 5), and the consensus human variable domain heavysubgroup III (huIII, SEQ ID NO: 6). The CDRs (H1, SEQ ID NO: 10; H2, SEQID NO: 11; and H3, SEQ ID NO: 12) are highlighted by underlining. Theresidue numbering is according to Kabat et al. (1991) supra. Thedifferences between the murine 9F3 and V13 sequences and the differencesbetween 9F3 and huIII sequences are indicated by asterisks.

FIG. 6 shows neutralization activity of the starting mAb 9F3 (leftpanel) and the chimeric protein CH8-2 (right panel) toward the viralgrowth inhibition exhibited by recombinant IFN-α subtypes in A549 cellschallenged with encephalomyocarditis (EMC) virus.

FIG. 7 depicts a model of humanized 9F3 version 13. Backbone of VL andVH domains is shown as a ribbon. CDRs are shown in white and are labeled(L1, L2, L3, H1, H2, H3). Framework side chains altered from human tomurine are shown in white and are labeled by residue number.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

A. Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. See, e.g. Singleton et al.,Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley &Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, N.Y.1989). For purposes of the present invention, the following terms aredefined below.

As used herein, the term “type I interferon” is defined to include allsubtypes of native sequence type I interferons of any mammalian species,including interferon-α, interferon-β, interferon-δ, interferon-ω andinterferon-τ. Similarly, the term “human type I interferon” is definedto include all subtypes of native sequence type I human interferons,including human interferon-α, interferon-β and interferon-ωclasses andwhich bind to a common cellular receptor.

Unless otherwise expressly provided, the terms “interferon-α,” “IFN-α,”and “human interferon-α”, “human IFN-α” and “hIFN-α” are used herein torefer to all species of native sequence human alpha interferons,including all subtypes of native sequence human interferons-α. Natural(native sequence) human interferon-α comprises 23 or more closelyrelated proteins encoded by distinct genes with a high degree ofstructural homology (Weissmann and Weber, Prog. Nucl. Acid. Res. Mol.Biol., 33: 251 [1986]; J. Interferon Res., 13: 443-444 [1993]; Robertset al., J. Interferon Cytokine Res. 18: 805-816 [1998]). The human IFN-αlocus comprises two subfamilies. The first subfamily consists of atleast 14 functional, non-allelic genes, including genes encoding IFN-αA(IFN-α2), IFN-αB (IFN-α8), IFN-α (IFN-α10), IFN-αD (IFN-α1), IFN-αE(IFN-α22), IFN-αF (IFN-α21), IFN-αG (IFN-α5), and IFN-αH (IFN-α14), andpseudogenes having at least 80% homology. The second subfamily, α_(II)or ω, contains at least 5 pseudogenes and one functional gene (denotedherein as “IFN-α_(II)1” or “IFN-ω”) which exhibits 70% homology with theIFN-α genes (Weissmann and Weber [1986] supra).

As used herein, the terms “first human interferon-α (hIFN-α) receptor”,“IFN-αR”, “hIFNAR1”, “IFNAR1”, and “Uze chain” are defined as the 557amino acid receptor protein cloned by Uze et al., Cell, 60: 225-234(1990), including an extracellular domain of 409 residues, atransmembrane domain of 21 residues, and an intracellular domain of 100residues, as shown in FIG. 5 on page 229 of Uze et al. Also encompassedby the foregoing terms are fragments of IFNAR1 that contain theextracellular domain (ECD) (or fragments of the ECD) of IFNAR1.

As used herein, the terms “second human interferon-α (hIFN-α) receptor”,“IFN-αβR”, “hIFNAR2”, “IFNAR2”, and “Novick chain” are defined as the515 amino acid receptor protein cloned by Domanski et al., J. Biol.Chem., 37: 21606-21611 (1995), including an extracellular domain of 217residues, a transmembrane domain of 21 residues, and an intracellulardomain of 250 residues, as shown in FIG. 1 on page 21608 of Domanski etal. Also encompassed by the foregoing terms are fragments of IFNAR2 thatcontain the extracellular domain (ECD) (or fragments of the ECD) ofIFNAR2, and soluble forms of IFNAR2, such as IFNAR2ECD fused to animmunoglobulin sequence, e.g. IFNAR2ECD-IgG Fc as described below.

The term “native sequence” in connection with type I interferon, IFN-αor any other polypeptide refers to a polypeptide that has the same aminoacid sequence as a corresponding polypeptide derived from nature,regardless of its mode of preparation. Such native sequence polypeptidecan be isolated from nature or can be produced by recombinant and/orsynthetic means or any combinations thereof. The term “native sequence”specifically encompasses naturally-occurring truncated or secreted forms(e.g., an extracellular domain sequence), naturally-occurring variantforms (e.g., alternatively spliced forms) and naturally-occurringallelic variants of the full length polypeptides.

“Polymerase chain reaction” or “PCR” refers to a procedure or techniquein which minute amounts of a specific piece of nucleic acid, RNA and/orDNA, are amplified as described in U.S. Pat. No. 4,683,195 issued 28Jul. 1987. Generally, sequence information from the ends of the regionof interest or beyond needs to be available, such that oligonucleotideprimers can be designed; these primers will be identical or similar insequence to opposite strands of the template to be amplified. The 5′terminal nucleotides of the two primers can coincide with the ends ofthe amplified material. PCR can be used to amplify specific RNAsequences, specific DNA sequences from total genomic DNA, and cDNAtranscribed from total cellular RNA, bacteriophage or plasmid sequences,etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol.51:263 (1987); Erlich, ed., PCR Technology (Stockton Press, NY, 1989).As used herein, PCR is considered to be one, but not the only, exampleof a nucleic acid polymerase reaction method for amplifying a nucleicacid test sample comprising the use of a known nucleic acid as a primerand a nucleic acid polymerase to amplify or generate a specific piece ofnucleic acid.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

“Native antibodies and immunoglobulins” are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (VH) followed by a number of constant domains. Eachlight chain has a variable domain at one end (VL) and a constant domainat its other end; the constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light- and heavy-chain variable domains (Chothia et al., J. Mol.Biol. 186:651 [1985]; Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A.82:4592 [1985]; Chothia et al., Nature 342: 877-883 [1989]).

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting a n-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al. (1991) supra). Theconstant domains are not involved directly in binding an antibody to anantigen, but exhibit various effector functions, such as participationof the antibody in antibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species,one heavy- and one light-chain variable domain can be covalently linkedby a flexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, called κand λ, based on the amino acid sequences of their constant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, εεγ, and μ respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The term “antibody” includes all classes and subclasses of intactimmunoglobulins. The term “antibody” also covers antibody fragments. Theterm “antibody” specifically covers monoclonal antibodies, includingantibody fragment clones.

“Antibody fragments” comprise a portion of an intact antibody thatcontains the antigen binding or variable region of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; single-chain antibody molecules, includingsingle-chain Fv (scFv) molecules; and multispecific antibodies formedfrom antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibody (orantibody fragment) obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Monoclonal antibodiesare highly specific, being directed against a single antigenic site.Furthermore, in contrast to conventional (polyclonal) antibodypreparations which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey are synthesized by the hybridoma culture, and are not contaminatedby other immunoglobulins. The modifier “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKohler et al., Nature, 256:495 (1975), or may be made by recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonalantibodies” also include clones of antigen-recognition and binding-sitecontaining antibody fragments (Fv clones) isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567 toCabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 [1984]).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from part or all of acomplementarity-determining region (CDR) of the recipient are replacedby residues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and optimizeantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature,321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988);Presta, Curr. Op. Struct. Biol., 2:593-596 (1992); and Clark, Immunol.Today 21: 397-402 (2000). The humanized antibody includes a Primatized™antibody wherein the antigen-binding region of the antibody is derivedfrom an antibody produced by immunizing macaque monkeys with the antigenof interest.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains, which enables the scFvto form the desired structure for antigen binding. For a review of scFvsee Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994),Dall'Acqua and Carter, Curr. Opin. Struct. Biol. 8: 443-450 (1998), andHudson, Curr. Opin. Immunol. 11: 548-557 (1999).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

By “neutralizing antibody” is meant an antibody molecule which is ableto eliminate or significantly reduce an effector function of a targetantigen to which it binds. Accordingly, a “neutralizing” anti-IFN-αantibody is capable of eliminating or significantly reducing an effectorfunction, such as receptor binding and/or elicitation of a cellularresponse, of IFN-α.

For the purpose of the present invention, the ability of an anti-IFN-αantibody to neutralize the receptor activation activity of IFN-α can bemonitored, for example, in a Kinase Receptor Activation (KIRA) Assay asdescribed in WO 95/14930, published Jun. 1, 1995, by measuring theability of a candidate antibody to reduce tyrosine phosphorylation(resulting from ligand binding) of the IFNAR1/R2 receptor complex.

For the purpose of the present invention, the ability of the anti-IFN-αantibodies to neutralize the elicitation of a cellular response by IFN-αis preferably tested by monitoring the neutralization of the antiviralactivity of IFN-α, as described by Kawade, J. Interferon Res. 1:61-70(1980), or Kawade and Watanabe, J. Interferon Res. 4:571-584 (1984), orYousefi, et al., Am. J. Clin. Pathol. 83: 735-740 (1985), or by testingthe ability of an anti-IFN-α antibody to neutralize the ability of IFN-αto activate the binding of the signaling molecule, interferon-stimulatedfactor 3 (ISGF3), to an oligonucleotide derived from theinterferon-stimulated response element (ISRE), in an electrophoreticmobility shift assay, as described by Kurabayashi et al., Mol. Cell.Biol., 15: 6386 (1995).

“Significant” reduction means at least about 60%, or at least about 70%,preferably at least about 75%, more preferably at least about 80%, evenmore preferably at least about 85%, still more preferably at least about90%, still more preferably at least about 95%, most preferably at leastabout 99% reduction of an effector function of the target antigen (e.g.IFN-α), such as receptor (e.g. IFNAR2) binding and/or elicitation of acellular response. Preferably, the “neutralizing” antibodies as definedherein will be capable of neutralizing at least about 60%, or at leastabout 70%, preferably at least about 75%, more preferably at least about80%, even more preferably at least about 85%, still more preferably atleast about 90%, still more preferably at least about 95%, mostpreferably at least about 99% of the anti-viral activity of IFN-α, asdetermined by the anti-viral assay of Kawade (1980), supra, or Yousefi(1985), supra. In another preferred embodiment, the “neutralizing”antibodies herein will be capable of reducing tyrosine phosphorylation,due to IFN-α binding, of the IFNAR1/IFNAR2 receptor complex, by at leastabout 60%, or at least about 70%, preferably at least about 75%, morepreferably at least about 80%; even more preferably at least about 85%,still more preferably at least about 90%, still more preferably at leastabout 95%, most preferably at least about 99%, as determined in the KIRAassay referenced above. In a particularly preferred embodiment, theneutralizing anti-IFN-α antibodies herein will be able to neutralizeall, or substantially all, subtypes of IFN-α and will not be able toneutralize IFN-β. In this context, the term “substantially all” meansthat the neutralizing anti-IFN-α antibody will neutralize at leastIFN-α1, IFN-α2, IFN-α4, IFN-α5, IFN-α8, IFN-α10, and IFN-α21.

For the purpose of the present invention, the ability of an anti-IFN-αantibody to block the binding of an IFN-α to receptor is defined as theproperty or capacity of a certain concentration of the antibody toreduce or eliminate the binding of IFN-α to IFNAR2 in a competitionbinding assay, as compared to the effect of an equivalent concentrationof irrelevant control antibody on IFN-α binding to IFNAR2 in the assay.Preferably, the blocking anti-IFN-α antibody reduces the binding ofIFN-α to IFNAR2 by at least about 50%, or at least about 55%, or atleast about 60%, or at least about 65%, or at least about 70%, or atleast about 75%, or at least about 80%, or at least about 85%, or atleast about 90%, or at least about 95%, or at least about 99%, ascompared to the irrelevant control antibody.

For the purpose of the present invention, the ability of an anti-IFN-αantibody to block the binding of IFN-α to IFNAR2 can be determined by aroutine competition assay such as that described in Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and DavidLane (1988). For example, the IFN-α-binding ELISA assay described inExample 2 below could be modified to employ competition binding betweenan anti-IFN-α antibody and a soluble IFNAR2. Such an assay could beperformed by layering the IFN-α on microtiter plates, incubating thelayered plates with serial dilutions of unlabeled anti-IFN-α antibody orunlabeled control antibody admixed with a selected concentration oflabeled IFNAR2ECD-human IgG Fc fusion protein, detecting and measuringthe signal in each incubation mixture, and then comparing the signalmeasurements exhibited by the various dilutions of antibody.

In a particularly preferred embodiment, the blocking anti-IFN-αantibodies herein will be able to block the IFNAR2-binding of all, orsubstantially all, subtypes of IFN-α and will not cross-react withIFN-β. In this context, the term “substantially all” means that theblocking anti-IFN-α antibody will block the IFNAR2-binding of at leastIFN-α1, IFN-α2, IFN-α4, IFN-α5, IFN-α8, IFN-α10, and IFN-α21. In aparticularly preferred embodiment, the blocking anti-IFN-α antibodies ofthe present invention will block the IFNAR2-binding of all knownsubtypes of IFN-α.

The term “epitope” is used to refer to binding sites for (monoclonal orpolyclonal) antibodies on protein antigens.

Antibodies which bind to a particular epitope can be identified by“epitope mapping.” There are many methods known in the art for mappingand characterizing the location of epitopes on proteins, includingsolving the crystal structure of an antibody-antigen complex,competition assays, gene fragment expression assays, and syntheticpeptide-based assays, as described, for example, in Chapter 11 of Harlowand Lane, Using Antibodies, a Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1999. Competition assays arediscussed above and below. According to the gene fragment expressionassays, the open reading frame encoding the protein is fragmented eitherrandomly or by specific genetic constructions and the reactivity of theexpressed fragments of the protein with the antibody to be tested isdetermined. The gene fragments may, for example, be produced by PCR andthen transcribed and translated into protein in vitro, in the presenceof radioactive amino acids. The binding of the antibody to theradioactively labeled protein fragments is then determined byimmunoprecipitation and gel electrophoresis. Certain epitopes can alsobe identified by using large libraries of random peptide sequencesdisplayed on the surface of phage particles (phage libraries).Alternatively, a defined library of overlapping peptide fragments can betested for binding to the test antibody in simple binding assays. Thelatter approach is suitable to define linear epitopes of about 5 to 15amino acids.

An antibody binds “essentially the same epitope” as a referenceantibody, when the two antibodies recognize identical or stericallyoverlapping epitopes. The most widely used and rapid methods fordetermining whether two epitopes bind to identical or stericallyoverlapping epitopes are competition assays, which can be configured inall number of different formats, using either labeled antigen or labeledantibody. Usually, the antigen is immobilized on a 96-well plate, andthe ability of unlabeled antibodies to block the binding of labeledantibodies is measured using radioactive or enzyme labels.

The term amino acid or amino acid residue, as used herein, refers tonaturally occurring L amino acids or to D amino acids as describedfurther below with respect to variants. The commonly used one- andthree-letter abbreviations for amino acids are used herein (BruceAlberts et al., Molecular Biology of the Cell, Garland Publishing, Inc.,New York (3d ed. 1994)).

“Percent (%) amino acid sequence identity” with respect to thepolypeptide sequences referred to herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in a sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign(DNASTAR) software. Those skilled in the art can determine appropriateparameters for measuring alignment, including any algorithms needed toachieve maximal alignment over the full-length of the sequences beingcompared. For purposes herein, however, % amino acid sequence identityvalues are obtained as described below by using the sequence comparisoncomputer program ALIGN-2. The ALIGN-2 sequence comparison computerprogram was authored by Genentech, Inc. and its source code has beenfiled with user documentation in the U.S. Copyright Office, WashingtonD.C., 20559, where it is registered under U.S. Copyright RegistrationNo. TXU510087. The ALIGN-2 program is publicly available throughGenentech, Inc., South San Francisco, Calif., and the source code forthe ALIGN-2 program and instructions for its use are disclosed inInternational Application Publication No. WO2000/39297 published Jul. 6,2000. The ALIGN-2 program should be compiled for use on a UNIX operatingsystem, preferably digital UNIX V4.0D. All sequence comparisonparameters are set by the ALIGN-2 program and do not vary.

For purposes herein, the % amino acid sequence identity of a given aminoacid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as describedabove using the ALIGN-2 sequence comparison computer program. However, %amino acid sequence identity may also be determined using the sequencecomparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res.25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may bedownloaded from http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses severalsearch parameters, wherein all of those search parameters are set todefault values including, for example, unmask=yes, strand=all, expectedoccurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program NCBI-BLAST2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “disease state” refers to a physiological state of a cell or ofa whole mammal in which an interruption, cessation, or disorder ofcellular or body functions, systems, or organs has occurred.

The term “effective amount” refers to an amount of a drug effective totreat (including prevention) of a disease, disorder or unwantedphysiological conditions in a mammal. In the present invention, an“effective amount” of an anti-IFN-α antibody may reduce, slow down ordelay an autoimmune disorder such as IDDM or SLE; reduce, prevent orinhibit (i.e., slow to some extent and preferably stop) the developmentof an autoimmune disorder such as IDDM or SLE; and/or relieve to someextent one or more of the symptoms associated with autoimmune disorderssuch as IDDM or SLE.

In the methods of the present invention, the term “control” andgrammatical variants thereof, are used to refer to the prevention,partial or complete inhibition, reduction, delay or slowing down of anunwanted event, e.g. physiological condition, such as the generation ofautoreactive T cells and development of autoimmunity.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those prone to have the disorder or thosein which the disorder is to be prevented. For purposes of thisinvention, beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

“Pharmaceutically acceptable” carriers, excipients, or stabilizers areones which are nontoxic to the cell or mammal being exposed thereto atthe dosages and concentrations employed. Often the physiologicallyacceptable carrier is an aqueous pH buffered solution. Examples ofphysiologically acceptable carriers include buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween™, polyethylene glycol (PEG), and Pluronics™.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

B. Methods for Carrying Out the Invention

1. Generation of Antibodies

(i) Polyclonal Antibodies

Methods of preparing polyclonal antibodies are known in the art.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. It may beuseful to conjugate the immunizing agent to a protein known to beimmunogenic in the mammal being immunized, such as serum albumin, orsoybean trypsin inhibitor. Examples of adjuvants which may be employedinclude Freund's complete adjuvant and MPL-TDM.

In another preferred embodiment, animals are immunized with a mixture ofvarious, preferably all, IFN-α subtypes in order to generate anti-IFN-αantibodies with broad reactivity against IFN-α subtypes. In anotherpreferred embodiment, animals are immunized with the mixture of humanIFN-α subtypes that is present in the human lymphoblastoid interferonssecreted by Burkitt lymphoma cells (Namalva cells) induced with Sendaivirus, as described in Example 1 below. A suitable preparation of suchhuman lymphoblastoid interferons can be obtained commercially (ProductNo. 1-9887) from Sigma Chemical Company, St. Louis, Mo.

(ii) Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature 256: 495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster or macaque monkey, is immunized as hereinabove described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103, [Academic Press,1996]).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOP-21 and MC.-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol. 133: 3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63,Marcel Dekker, Inc., New York, [1987]).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.107: 220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the cells may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103,Academic Press, 1996). Suitable culture media for this purpose include,for example, DMEM or RPMI-1640 medium. In addition, the hybridoma cellsmay be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells. The DNA also may be modified, for example, by substituting thecoding sequence for human heavy and light chain constant domains inplace of the homologous murine sequences (Morrison, et al., Proc. Nat.Acad. Sci. 81: 6851 [1984]), or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In that manner, “chimeric” or “hybrid”antibodies are prepared that have the binding specificity of ananti-IFN-α monoclonal antibody herein.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for an IFN-αand another antigen-combining site having specificity for a differentantigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

Recombinant production of antibodies will be described in more detailbelow.

(iii) Humanized Antibodies

Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a non-human source. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers by substitutingrodent CDRs or CDR sequences for the corresponding sequences of a humanantibody (Jones et al., Nature 321: 522-525 [1986]; Riechmann et al.,Nature 332: 323-327 [1988]; Verhoeyen et al., Science 239: 1534-1536[1988)]; reviewed in Clark, Immunol. Today 21: 397-402 [2000]).

Accordingly, such “humanized” antibodies are chimeric antibodies(Cabilly, supra), wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possibly some FRresidues are substituted by residues from analogous sites in rodentantibodies.

It is important that antibodies be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, according to a preferred method, humanized antibodiesare prepared by a process of analysis of the parental sequences andvarious conceptual humanized products using three-dimensional models ofthe parental and humanized sequences. Three dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e. the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from theconsensus and import sequence so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the CDR residues are directly and mostsubstantially involved in influencing antigen binding. For furtherdetails, see U.S. Pat. No. 5,821,337.

(iv) Human Antibodies

Attempts to use the same technology for generating human mAbs have beenhampered by the lack of a suitable human myeloma cell line. The bestresults were obtained using heteromyelomas (mouse× human hybridmyelomas) as fusion partners (Kozbor, J. Immunol. 133: 3001 (1984);Brodeur, et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63, Marcel Dekker, Inc., New York, 1987).Alternatively, human antibody-secreting cells can be immortalized byinfection with the Epstein-Barr virus (EBV). However, EBV-infected cellsare difficult to clone and usually produce only relatively low yields ofimmunoglobulin (James and Bell, J. Immunol. Methods 100: 5-40 [1987]).In future, the immortalization of human B cells might possibly beachieved by introducing a defined combination of transforming genes.Such a possibility is highlighted by a recent demonstration that theexpression of the telomerase catalytic subunit together with the SV40large T oncoprotein and an oncogenic allele of H-ras resulted in thetumorigenic conversion of normal human epithelial and fibroblast cells(Hahn et al., Nature 400: 464-468 [1999]).

It is now possible to produce transgenic animals (e.g. mice) that arecapable, upon immunization, of producing a repertoire of humanantibodies in the absence of endogenous immunoglobulin production(Jakobovits et al., Nature 362: 255-258 [1993]; Lonberg and Huszar, Int.Rev. Immunol. 13: 65-93 [1995]; Fishwild et al., Nat. Biotechnol. 14:845-851 [1996]; Mendez et al., Nat. Genet. 15: 146-156 [1997]; Green, J.Immunol. Methods 231: 11-23 [1999]; Tomizuka et al., Proc. Natl. Acad.Sci. USA 97: 722-727 [2000]; reviewed in Little et al., Immunol. Today21: 364-370 [2000]). For example, it has been described that thehomozygous deletion of the antibody heavy chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production (Jakobovits et al., Proc.Natl. Acad. Sci. USA 90: 2551-2555 [1993]). Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant miceresults in the production of human antibodies upon antigen challenge(Jakobovits et al., Nature 362: 255-258 [1993]).

Mendez et al. (Nature Genetics 15: 146-156 [1997]) have generated a lineof transgenic mice designated as “XenoMouse® II” that, when challengedwith an antigen, generates high affinity fully human antibodies. Thiswas achieved by germ-line integration of megabase human heavy chain andlight chain loci into mice with deletion into endogenous J_(H) segmentas described above. The XenoMouse® II harbors 1,020 kb of human heavychain locus containing approximately 66 V_(H) genes, complete D_(H) andJ_(H) regions and three different constant regions (μ, δ and γ), andalso harbors 800 kb of human κ locus containing 32 Vκ genes, Jκ segmentsand Cκ genes. The antibodies produced in these mice closely resemblethat seen in humans in all respects, including gene rearrangement,assembly, and repertoire. The human antibodies are preferentiallyexpressed over endogenous antibodies due to deletion in endogenous J_(H)segment that prevents gene rearrangement in the murine locus.

Tomizuka et al. (Proc. Natl. Acad. Sci. USA 97: 722-727 [2000]) haverecently described generation of a double trans-chromosomic (Tc) mice byintroducing two individual human chromosome fragments (hCFs), onecontaining the entire Ig heavy chain locus (IgH, ˜1.5 Mb) and the otherthe entire κ light chain locus (Igκ, ˜2 Mb) into a mouse strain whoseendogenous IgH and Igκ loci were inactivated. These mice mountedantigen-specific human antibody response in the absence of mouseantibodies. The Tc technology may allow for the humanization of overmegabase-sized, complex loci or gene clusters (such as those encodingT-cell receptors, major histocompatibility complex, P450 cluster etc) inmice or other animals. Another advantage of the method is theelimination of a need of cloning the large loci. This is a significantadvantage since the cloning of over megabase-sized DNA fragmentsencompassing whole Ig loci remains difficult even with the use of yeastartificial chromosomes (Peterson et al., Trends Genet. 13: 61-66 [1997];Jacobovits, Curr. Biol. 4: 761-763 [1994]). Moreover, the constantregion of the human IgH locus is known to contain sequences difficult toclone (Kang and Cox, Genomics 35: 189-195 [1996]).

Alternatively, the phage display technology can be used to produce humanantibodies and antibody fragments in vitro, from immunoglobulin variable(V) domain gene repertoires from unimmunized donors (McCafferty et al.,Nature 348: 552-553 [1990]; reviewed in Kipriyanov and Little, Mol.Biotechnol. 12: 173-201 [1999]; Hoogenboom and Chames, Immunol. Today21: 371-378 [2000]). According to this technique, antibody V domaingenes are cloned in-frame into either a major or minor coat protein geneof a filamentous bacteriophage, such as M13 or fd, and displayed asfunctional antibody fragments on the surface of the phage particle.Because the filamentous particle contains a single-stranded DNA copy ofthe phage genome, selections based on the functional properties of theantibody also result in selection of the gene encoding the antibodyexhibiting those properties. Thus, the phage mimics some of theproperties of the B-cell. Phage display can be performed in a variety offormats (reviewed in Johnson and Chiswell, Current Opinion in StructuralBiology 3: 564-571 [1993)]; Winter et al., Annu. Rev. Immunol. 12:433-455 [1994]; Dall'Acqua and Carter, Curr. Opin. Struct. Biol. 8:443-450 [1998]; Hoogenboom and Chames, Immunol. Today 21: 371-378[2000]). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., (Nature 352: 624-628 [1991]) isolated adiverse array of anti-oxazolone antibodies from a small randomcombinatorial library of V genes derived from the spleens of immunizedmice. A repertoire of V genes from unimmunized human donors can beconstructed and antibodies to a diverse array of antigens (includingself-antigens) can be isolated essentially following the techniquesdescribed by Marks et al., J. Mol. Biol. 222: 581-597 (1991), orGriffiths et al., EMBO J. 12: 725-734 (1993). In a natural immuneresponse, antibody genes accumulate mutations at a high rate (somatichypermutation). Some of the changes introduced will confer higheraffinity, and B cells displaying high-affinity surface immunoglobulinare preferentially replicated and differentiated during subsequentantigen challenge. This natural process can be mimicked by employing thetechnique known as “chain shuffling” (Marks et al., Bio/Technol. 10:779-783 [1992]). In this method, the affinity of “primary” humanantibodies obtained by phage display can be improved by sequentiallyreplacing the heavy and light chain V region genes with repertoires ofnaturally occurring variants (repertoires) of V domain genes obtainedfrom unimmunized donors. This technique allows the production ofantibodies and antibody fragments with affinities in the nM range. Astrategy for making very large phage antibody repertoires (also known as“the mother-of-all libraries”) has been described by Waterhouse et al.,Nucl. Acids Res. 21: 2265-2266 (1993), and the isolation of a highaffinity human antibody directly from such large phage library isreported by Griffiths et al., EMBO J. 13: 3245-3260 (1994). Geneshuffling can also be used to derive human antibodies from rodentantibodies, where the human antibody has similar affinities andspecificities to the starting rodent antibody. According to this method,which is also referred to as “epitope imprinting”, the heavy or lightchain V domain gene of rodent antibodies obtained by phage displaytechnique is replaced with a repertoire of human V domain genes,creating rodent-human chimeras. Selection on antigen results inisolation of human variable capable of restoring a functionalantigen-binding site, i.e. the epitope governs (imprints) the choice ofpartner. When the process is repeated in order to replace the remainingrodent V domain, a human antibody is obtained (see PCT patentapplication WO 93/06213, published 1 Apr. 1993). Unlike traditionalhumanization of rodent antibodies by CDR grafting, this techniqueprovides completely human antibodies, which have no framework or CDRresidues of rodent origin.

(v) Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is foran IFN-α to provide a neutralizing antibody, the other one is for anyother antigen.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities(Millstein and Cuello, Nature 305: 537-539 [1983]). Because of therandom assortment of immunoglobulin heavy and light chains, thesehybridomas (quadromas) produce a potential mixture of 10 differentantibody molecules, of which only one has the correct bispecificstructure. The purification of the correct molecule, which is usuallydone by affinity chromatography steps, is rather cumbersome, and theproduct yields are low. Similar procedures are disclosed in PCTapplication publication No. WO 93/08829 (published 13 May 1993), and inTraunecker et al., EMBO J. 10: 3655-3659 (1991).

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, CH₂ and CH3 regions. Itis preferred to have the first heavy chain constant region (CH₁)containing the site necessary for light chain binding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are co-transfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance. In a preferred embodiment of this approach,the bispecific antibodies are composed of a hybrid immunoglobulin heavychain with a first binding specificity in one arm, and a hybridimmunoglobulin heavy chain-light chain pair (providing a second bindingspecificity) in the other arm. It was found that this asymmetricstructure facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations, as the presence of animmunoglobulin light chain in only one half of the bispecific moleculeprovides for a facile way of separation.

For further details of generating bispecific antibodies see, forexample, Suresh et al., Methods in Enzymology 121, 210 (1986).

(vi) Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (PCT application publication Nos. WO91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

(vii) Antibody Fragments

In certain embodiments, the neutralizing anti-IFN-α antibody (includingmurine, human and humanized antibodies, and antibody variants) is anantibody fragment. Various techniques have been developed for theproduction of antibody fragments. Traditionally, these fragments werederived via proteolytic digestion of intact antibodies (see, e.g.,Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 [1992] andBrennan et al., Science 229:81 [1985]). However, these fragments can nowbe produced directly by recombinant host cells (reviewed in Hudson,Curr. Opin. Immunol. 11: 548-557 [1999]; Little et al., Immunol. Today21: 364-370 [2000]). For example, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 [1992]). In anotherembodiment, the F(ab′)₂ is formed using the leucine zipper GCN4 topromote assembly of the F(ab′)₂ molecule. According to another approach,Fv, Fab or F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Other techniques for the production of antibodyfragments will be apparent to the skilled practitioner.

(viii) Amino Acid Sequence Variants of Antibodies

Amino acid sequence modification(s) of the anti-IFN-α antibodiesdescribed herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of the anti-IFN-α antibodies areprepared by introducing appropriate nucleotide changes into the nucleicacid encoding the anti-IFN-α antibody chains, or by peptide synthesis.Such modifications include, for example, deletions from, and/orinsertions into and/or substitutions of, residues within the amino acidsequences of the anti-IFN-α antibody. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid changes also may alter post-translational processes ofthe anti-IFN-α antibody, such as changing the number or position ofglycosylation sites.

A useful method for identification of certain residues or regions of theneutralizing anti-IFN-α antibody that are preferred locations formutagenesis is called “alanine scanning mutagenesis,” as described byCunningham and Wells Science, 244:1081-1085 (1989). Here, a residue orgroup of target residues are identified (e.g., charged residues such asarg, asp, his, lys, and glu) and replaced by a neutral or negativelycharged amino acid (most preferably alanine or polyalanine) to affectthe interaction of the amino acids with the antigen. Those amino acidlocations demonstrating functional sensitivity to the substitutions thenare refined by introducing further or other variants at, or for, thesites of substitution. Thus, while the site for introducing an aminoacid sequence variation is predetermined, the nature of the mutation perse need not be predetermined. For example, to analyze the performance ofa mutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed anti-IFN-αantibody variants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intra-sequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean anti-IFN-α neutralizing antibody with an N-terminal methionyl residueor the antibody fused to an epitope tag. Other insertional variants ofthe anti-IFN-α antibody molecule include the fusion to the N- orC-terminus of the antibody of an enzyme or a polypeptide which increasesthe serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the neutralizinganti-IFN-α antibody molecule removed and a different residue inserted inits place. The sites of greatest interest for substitution mutagenesisinclude the hypervariable regions, but FR alterations are alsocontemplated. Conservative substitutions are shown in Table 1 under theheading of “preferred substitutions”. If such substitutions result in achange in biological activity, then more substantial changes,denominated “exemplary substitutions” in Table 1, or as furtherdescribed below in reference to amino acid classes, may be introducedand the products screened.

TABLE 1 Exemplary Preferred Original Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;asp, lys; arg gln Asp (D) glu; asn glu Cys (C) ser; ala ser Gln (Q) asn;glu asn Glu (E) asp; gln asp Gly (G) ala ala His (H) asn; gln; lys; argarg Ile (I) leu; val; met; ala; leu phe; norleucine Leu (L) norleucine;ile; val; ile met; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe;ile leu Phe (F) leu; val; ile; ala; tyr tyr Pro (P) ala ala Ser (S) thrthr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser pheVal (V) ile; leu; met; phe; leu ala; norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the neutralizing anti-IFN-α antibody also may be substituted,generally with serine, to improve the oxidative stability of themolecule and prevent aberrant crosslinking. Conversely, cysteine bond(s)may be added to the antibody to improve its stability (particularlywhere the antibody is an antibody fragment such as a Fv fragment).

A particularly preferred type of substitution variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionvariants is affinity maturation using phage display. Briefly, severalhypervariable region sites (e.g. 6-7 sites) are mutated to generate allpossible amino substitutions at each site. The antibody variants thusgenerated are displayed in a monovalent fashion from filamentous phageparticles as fusions to the gene III product of M13 packaged within eachparticle. The phage-displayed variants are then screened for theirbiological activity (e.g. antagonist activity) as herein disclosed. Inorder to identify candidate hypervariable region sites for modification,alanine scanning mutagenesis can be performed to identify hypervariableregion residues contributing significantly to antigen binding.Alternatively, or in addition, it may be beneficial to analyze a crystalstructure of the antigen-antibody complex to identify contact pointsbetween the antibody and IFN-α. Such contact residues and neighboringresidues are candidates for substitution according to the techniqueselaborated herein. Once such variants are generated, the panel ofvariants is subjected to screening as described herein and antibodieswith superior properties in one or more relevant assays may be selectedfor further development.

(ix) Glycosylation Variants

Antibodies are glycosylated at conserved positions in their constantregions (Jefferis and Lund, Chem. Immunol. 65:111-128 [1997]; Wright andMorrison, Trends Biotechnol. 15:26-32 [1997]). The oligosaccharide sidechains of the immunoglobulins affect the protein's function (Boyd etal., Mol. Immunol. 32:1311-1318 [1996]; Wittwe and Howard, Biochem.29:4175-4180 [1990]), and the intramolecular interaction betweenportions of the glycoprotein which can affect the conformation andpresented three-dimensional surface of the glycoprotein (Jefferis andLund, supra; Wyss and Wagner, Current Opin. Biotech. 7:409-416 [1996]).Oligosaccharides may also serve to target a given glycoprotein tocertain molecules based upon specific recognition structures. Forexample, it has been reported that in agalactosylated IgG, theoligosaccharide moiety ‘flips’ out of the inter-CH2 space and terminalN-acetylglucosamine residues become available to bind mannose bindingprotein (Malhotra et al., Nature Med. 1:237-243 [1995]). Removal byglycopeptidase of the oligosaccharides from CAMPATH-1H (a recombinanthumanized murine monoclonal IgG1 antibody which recognizes the CDw52antigen of human lymphocytes) produced in Chinese Hamster Ovary (CHO)cells resulted in a complete reduction in complement mediated lysis(CMCL) (Boyd et al., Mol. Immunol. 32:1311-1318 [1996]), while selectiveremoval of sialic acid residues using neuraminidase resulted in no lossof DMCL. Glycosylation of antibodies has also been reported to affectantibody-dependent cellular cytotoxicity (ADCC). In particular, CHOcells with tetracycline-regulated expression ofβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing formation of bisecting GlcNAc, wasreported to have improved ADCC activity (Umana et al., Mature Biotech.17:176-180 [1999]).

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Glycosylation variants of antibodies are variants in which theglycosylation pattern of an antibody is altered. By altering is meantdeleting one or more carbohydrate moieties found in the antibody, addingone or more carbohydrate moieties to the antibody, changing thecomposition of glycosylation (glycosylation pattern), the extent ofglycosylation, etc.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).Similarly, removal of glycosylation sites can be accomplished by aminoacid alteration within the native glycosylation sites of the antibody.

The amino acid sequence is usually altered by altering the underlyingnucleic acid sequence. Nucleic acid molecules encoding amino acidsequence variants of the anti-IFN-α antibody are prepared by a varietyof methods known in the art. These methods include, but are not limitedto, isolation from a natural source (in the case of naturally occurringamino acid sequence variants) or preparation by oligonucleotide-mediated(or site-directed) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared variant or a non-variant version ofthe anti-IFN-α antibody.

The glycosylation (including glycosylation pattern) of antibodies mayalso be altered without altering the amino acid sequence or theunderlying nucleotide sequence. Glycosylation largely depends on thehost cell used to express the antibody. Since the cell type used forexpression of recombinant glycoproteins, e.g. antibodies, as potentialtherapeutics is rarely the native cell, significant variations in theglycosylation pattern of the antibodies can be expected (see, e.g. Hseet al., J. Biol. Chem. 272:9062-9070 [1997]). In addition to the choiceof host cells, factors which affect glycosylation during recombinantproduction of antibodies include growth mode, media formulation, culturedensity, oxygenation, pH, purification schemes and the like. Variousmethods have been proposed to alter the glycosylation pattern achievedin a particular host organism including introducing or overexpressingcertain enzymes involved in oligosaccharide production (U.S. Pat. Nos.5,047,335; 5,510,261 and 5.278,299). Glycosylation, or certain types ofglycosylation, can be enzymatically removed from the glycoprotein, forexample using endoglycosidase H (Endo H). In addition, the recombinanthost cell can be genetically engineered, e.g. make defective inprocessing certain types of polysaccharides. These and similartechniques are well known in the art.

The glycosylation structure of antibodies can be readily analyzed byconventional techniques of carbohydrate analysis, including lectinchromatography, NMR, Mass spectrometry, HPLC, GPC, monosaccharidecompositional analysis, sequential enzymatic digestion, and HPAEC-PAD,which uses high pH anion exchange chromatography to separateoligosaccharides based on charge. Methods for releasing oligosaccharidesfor analytical purposes are also known, and include, without limitation,enzymatic treatment (commonly performed using peptide-N-glycosidaseFiendo-β-galactosidase), elimination using harsh alkaline environment torelease mainly O-linked structures, and chemical methods using anhydroushydrazine to release both N- and O-linked oligosaccharides.

(x) Other Modifications of Antibodies

The neutralizing anti-IFN-α antibodies disclosed herein may also beformulated as immunoliposomes. Liposomes containing the antibody areprepared by methods known in the art, such as described in Epstein etal., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc.Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and4,544,545. Liposomes with enhanced circulation time are disclosed inU.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257:286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.81(19):1484 (1989).

The antibody of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g., a peptidyl chemotherapeutic agent, see WO81/01145) to anactive drug. See, for example, WO 88/07378 and U.S. Pat. No. 4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active form exhibiting the desired biologicalproperties.

Enzymes that are useful in the method of this invention include, but arenot limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as “abzymes”, can be used to convert the prodrugs ofthe invention into free active drugs (see, e.g., Massey, Nature328:457-458 (1987)). Antibody-abzyme conjugates can be prepared asdescribed herein for delivery of the abzyme to a desired cellpopulation.

The enzymes can be covalently bound to the neutralizing anti-IFN-αantibodies by techniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the invention linked to at least a functionally activeportion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature 312:604-608 [1984]).

In certain embodiments of the invention, it may be desirable to use anantibody fragment, rather than an intact antibody. In this case, it maybe desirable to modify the antibody fragment in order to increase itsserum half-life. This may be achieved, for example, by incorporation ofa salvage receptor binding epitope into the antibody fragment (e.g., bymutation of the appropriate region in the antibody fragment or byincorporating the epitope into a peptide tag that is then fused to theantibody fragment at either end or in the middle, e.g., by DNA orpeptide synthesis). See WO96/32478 published Oct. 17, 1996.

The salvage receptor binding epitope generally constitutes a regionwherein any one or more amino acid residues from one or two loops of aFc domain are transferred to an analogous position of the antibodyfragment. Even more preferably, three or more residues from one or twoloops of the Fc domain are transferred. Still more preferred, theepitope is taken from the CH2 domain of the Fc region (e.g., of an IgG)and transferred to the CH1, CH3, or V_(H) region, or more than one suchregion, of the antibody. Alternatively, the epitope is taken from theCH2 domain of the Fc region and transferred to the C_(L) region or V_(L)region, or both, of the antibody fragment.

Covalent modifications of the neutralizing anti-IFN-α antibodies arealso included within the scope of this invention. They may be made bychemical synthesis or by enzymatic or chemical cleavage of the antibody,if applicable. Other types of covalent modifications of the antibody areintroduced into the molecule by reacting targeted amino acid residues ofthe antibody with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues.Exemplary covalent modifications of polypeptides are described in U.S.Pat. No. 5,534,615, specifically incorporated herein by reference. Apreferred type of covalent modification of the antibody compriseslinking the antibody to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

2. Screening for Antibodies with the Desired Properties

Techniques for generating antibodies have been described above.Anti-IFN-α antibodies with the desired, broad range neutralizingproperties can then be identified by methods known in the art.

(1) Binding Assays

Thus, for example, the neutralizing anti-IFN-α antibodies of the presentinvention can be identified in IFN-α binding assays, by incubating acandidate antibody with one or more individual IFN-α subtypes, or anarray or mixture of various IFN-α subtypes, and monitoring binding andneutralization of a biological activity of IFN-α. The binding assay maybe performed with purified IFN-α, polypeptide(s). In one embodiment, thebinding assay is a competitive binding assay, where the ability of acandidate antibody to compete with a known anti-IFN-α antibody for IFN-αbinding is evaluated. The assay may be performed in various formats,including the ELISA format, also illustrated in the Examples below.IFN-α binding of a candidate antibody may also be monitored in aBIAcore™ Biosensor assay, as described below.

Any suitable competition binding assay known in the art can be used tocharacterize the ability of a candidate anti-IFN-α monoclonal antibodyto compete with murine anti-human IFN-α monoclonal antibody 9F3 forbinding to a particular IFN-α species. A routine competition assay isdescribed in Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988). In another embodiment, theIFN-α-binding ELISA assay described in Example 2 below could be modifiedto employ IFN-α binding competition between a candidate antibody and the9F3 antibody. Such an assay could be performed by layering the IFN-α onmicrotiter plates, incubating the layered plates with serial dilutionsof unlabeled anti-IFN-α antibody or unlabeled control antibody admixedwith a selected concentration of labeled 9F3 antibody, detecting andmeasuring the signal from the 9F3 antibody label, and then comparing thesignal measurements exhibited by the various dilutions of antibody.

(ii) Antiviral Assays

The ability of a candidate antibody to neutralize a biological activityof IFN-α can, for example, be carried out by monitoring theneutralization of the antiviral activity of IFN-α as described byKawade, J. Interferon Res. 1:61-70 (1980), or Kawade and Watanabe, J.Interferon Res. 4:571-584 (1984). Briefly, a fixed concentration ofIFN-α premixed with various dilutions of a candidate antibody is addedto human amnion-derived FL cells, and the ability of the candidateantibody to neutralize the antiviral activity of IFN-α is determined,using an appropriate virus, e.g. Sindbis virus. The titers are expressedin international units (IU), as determined with the internationalreference human IFN-α (NIH Ga23-901-527).

The candidate anti-IFN-α antibody is considered able to inhibit theanti-viral activity of a selected IFN-α subtype if a certainconcentration of the antibody inhibits more anti-viral activity than thebaseline level of anti-viral activity inhibition measured in thepresence of an equivalent concentration of control antibody. Optionally,the certain concentration of the candidate anti-IFN-α, antibody willinhibit at least or about 60%, or at least or about 70%, preferably atleast or about 75%, or more preferably at least or about 80%, or evenmore preferably at least or about 85%, or still more preferably at leastor about 90%, or still more preferably at least or about 95%, or mostpreferably at least or about 99% of the anti-viral activity of theselected IFN-α subtype in the anti-viral activity assay as compared tobaseline activity measured in the presence of an equivalentconcentration of control antibody. The candidate anti-IFN-α antibody isconsidered unable to inhibit the anti-viral activity of a selected IFN-αsubtype if there is no concentration of the antibody that exhibits moreanti-viral activity inhibition than the baseline level of anti-viralactivity inhibition measured in the presence of an equivalentconcentration of control antibody.

In a preferred embodiment, each interferon species used in the viralinfectivity assay is titrated to a concentration that provides the samelevel of inhibition of viral growth as that induced by a preselectednumber of units of an IFN-α standard. This concentration serves toprovide the normalized units of the subject interferon species. In orderto assess the ability of an anti-IFN-α antibody to inhibit theanti-viral activity of various IFN-α subtypes, the effectiveconcentration (EC50) of anti-IFN-α antibody for inhibiting 50% of aparticular IFN-α subtype's anti-viral activity (at the concentrationtitrated to provide the normalized units of activity) is determined foreach IFN-α subtype to be tested.

In one embodiment, the antiviral activity neutralization assay isperformed as described in Example 1 below. Briefly, A549 cells are grownto a density of 5×10⁵ A549 cells/well on 96-well microtiter plates.Serial dilutions of candidate anti-IFN-α antibody are incubated with 0.2units/μl of a selected IFN-α subtype (normalized to 0.2 units/μl of NIHreference standard recombinant human IFN-α2) in a total volume of 100 μlat 37° C. for one hour. Each 100 μl volume of antibody/interferonincubation mixture is then added to 5×10⁵ A549 cells and 100 μl ofculture medium in an individual well on the microtiter plate, yielding afinal IFN-α concentration of 100 units/ml in each well. The resultingcell culture mixtures are incubated for 24 hours at 37° C. Cells arethen challenged with 2×10⁵ pfu of encephalomyocarditis (EMC) virus andincubated for an additional 24 hours at 37° C. At the end of theincubation, cell viability is determined by visual microscopicexamination or crystal violet staining. The effective concentration(EC50) of a candidate IFN-α antibody for inhibiting 50% of a particularIFN-α subtype's anti-viral activity in the assay is determined for eachIFN-α subtype to be tested.

In one aspect of the invention, the anti-IFN-α antibody that exhibitsanti-viral activity neutralization against the subject IFN-α subtypeswill exhibit an EC50 of up to or about 20 μg/ml, or up to or about 10μg/ml, or up to or about 5 μg/ml, or up to or about 4 μg/ml, or up to orabout 3 μg/ml, or up to or about 2 μg/ml, or up to or about 1 μg/ml withrespect to each of the subject IFN-α subtypes in the A549 cell EMC viralinhibition assay described above.

In another aspect of the invention, the anti-IFN-α antibody thatexhibits anti-viral activity neutralization against the subject IFN-αsubtypes will exhibit an EC50 from or about 0.1 μg/ml to or about 20μg/ml, from or about 0.1 μg/ml to or about 10 μg/ml, from or about 0.1μg/ml to or about 5 μg/ml, or from or about 0.1 μg/ml to or about 4μg/ml, from or about 0.1 μg/ml to or about 3 μg/ml, from or about 0.1μg/ml to or about 2 μg/ml, or from or about 0.1 μg/ml to or about 1μg/ml with respect to each of the subject IFN-α subtypes in the A549cell EMC viral inhibition assay described above.

In another aspect of the invention, the anti-IFN-α antibody thatanti-viral activity neutralization against the subject IFN-α subtypeswill exhibit an EC50 from or about 0.2 μg/ml to or about 20 μg/ml, fromor about 0.2 μg/ml to or about 10 μg/ml, from or about 0.2 μg/ml to orabout 5 μg/ml, or from or about 0.2 μg/ml to or about 4 μg/ml, from orabout 0.2 μg/ml to or about 3 μg/ml, from or about 0.2 μg/ml to or about2 μg/ml, or from or about 0.2 μg/ml to or about 1 μg/ml with respect toeach of the subject IFN-α subtypes in the A549 cell EMC viral inhibitionassay described above.

In another aspect of the invention, the anti-IFN-α antibody thatneutralizes the anti-viral activity of the subject IFN-α subtypes willexhibit an EC50 from or about 0.3 μg/ml to or about 20 μg/ml, from orabout 0.3 μg/ml to or about 10 μg/ml, from or about 0.3 μg/ml to orabout 5 μg/ml, or from or about 0.3 μg/ml to or about 4 μg/ml, from orabout 0.3 μg/ml to or about 3 μg/ml, from or about 0.3 μg/ml to or about2 μg/ml, or from or about 0.3 μg/ml to or about 1 μg/ml with respect toeach of the subject IFN-α subtypes in the A549 cell EMC viral inhibitionassay described above.

In another aspect of the invention, the anti-IFN-α antibody thatneutralizes the anti-viral activity of the subject IFN-α subtypes willexhibit an EC50 from or about 0.4 μg/ml to or about 20 μg/ml, from orabout 0.4 μg/ml to or about 10 μg/ml, from or about 0.4 μg/ml to orabout 5 μg/ml, or from or about 0.4 μg/ml to or about 4 μg/ml, from orabout 0.4 μg/ml to or about 3 μg/ml, from or about 0.4 μg/ml to or about2 μg/ml, or from or about 0.4 μg/ml to or about 1 μg/ml with respect toeach of the subject IFN-α subtypes in the A549 cell EMC viral inhibitionassay described above.

In another aspect of the invention, the anti-IFN-α antibody thatneutralizes the anti-viral activity of the subject IFN-α subtypes willexhibit an EC50 from or about 0.5 μg/ml to or about 20 μg/ml, from orabout 0.5 μg/ml to or about 10 μg/ml, from or about 0.5 μg/ml to orabout 5 μg/ml, or from or about 0.5 μg/ml to or about 4 μg/ml, from orabout 0.5 μg/ml to or about 3 μg/ml, from or about 0.5 μg/ml to or about2 μg/ml, or from or about 0.5 μg/ml to or about 1 μg/ml with respect toeach of the subject IFN-α subtypes in the A549 cell EMC viral inhibitionassay described above.

In another aspect of the invention, the anti-IFN-α antibody thatneutralizes the anti-viral activity of the subject IFN-α subtypes willexhibit an EC50 from or about 0.6 μg/ml to or about 20 μg/ml, from orabout 0.6 μg/ml to or about 10 μg/ml, from or about 0.6 μg/ml to orabout 5 μg/ml, or from or about 0.6 μg/ml to or about 4 μg/ml, from orabout 0.6 μg/ml to or about 3 μg/ml, from or about 0.6 μg/ml to or about2 μg/ml, or from or about 0.6 μg/ml to or about 1 μg/ml with respect toeach of the subject IFN-α subtypes in the A549 cell EMC viral inhibitionassay described above.

In another aspect of the invention, the anti-IFN-α antibody thatneutralizes the anti-viral activity of the subject IFN-α subtypes willexhibit an EC50 from or about 0.7 μg/ml to or about 20 μg/ml, from orabout 0.7 μg/ml to or about 10 μg/ml, from or about 0.7 μg/ml to orabout 5 μg/ml, or from or about 0.7 μg/ml to or about 4 μg/ml, from orabout 0.7 μg/ml to or about 3 μg/ml, from or about 0.7 μg/ml to or about2 μg/ml, or from or about 0.7 μg/ml to or about 1 μg/ml with respect toeach of the subject IFN-α subtypes in the A549 cell EMC viral inhibitionassay described above.

In another aspect of the invention, the anti-IFN-α antibody thatneutralizes the anti-viral activity of the subject IFN-α subtypes willexhibit an EC50 from or about 0.8 μg/ml to or about 20 μg/ml, from orabout 0.8 μg/ml to or about 10 μg/ml, from or about 0.8 μg/ml to orabout 5 μg/ml, or from or about 0.8 μg/ml to or about 4 μg/ml, from orabout 0.8 μg/ml to or about 3 μg/ml, from or about 0.8 μg/ml to or about2 μg/ml, or from or about 0.8 μg/ml to or about 1 μg/ml with respect toeach of the subject IFN-α subtypes in the A549 cell EMC viral inhibitionassay described above.

In another aspect of the invention, the anti-IFN-α antibody thatneutralizes the anti-viral activity of the subject IFN-α subtypes willexhibit an EC50 from or about 0.9 μg/ml to or about 20 μg/ml, from orabout 0.9 μg/ml to or about 10 μg/ml, from or about 0.9 μg/ml to orabout 5 μg/ml, or from or about 0.9 μg/ml to or about 4 μg/ml, from orabout 0.9 μg/ml to or about 3 μg/ml, from or about 0.9 μg/ml to or about2 μg/ml, or from or about 0.9 μg/ml to or about 1 μg/ml with respect toeach of the subject IFN-α subtypes in the A549 cell EMC viral inhibitionassay described above.

In another aspect of the invention, the anti-IFN-α antibody thatneutralizes the anti-viral activity of the subject IFN-α subtypes willexhibit an EC50 from or about 1 μg/ml to or about 20 μg/ml, from orabout 1 μg/ml to or about 10 μg/ml, from or about 1 μg/ml to or about 5μg/ml, or from or about 1 μg/ml to or about 4 μg/ml, from or about 1μg/ml to or about 3 μg/ml, or from or about 1 μg/ml to or about 2 μg/mlwith respect to each of the subject IFN-α subtypes in the A549 cell EMCviral inhibition assay described above.

(iii) Cross-Blocking Assays

To screen for antibodies which bind to an epitope on IFN-α bound by anantibody of interest, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, or additionally, epitope mapping can be performed bymethods known in the art. For example, the IFN-α epitope bound by amonoclonal antibody of the present invention can be determined bycompetitive binding analysis as described in Fendly et al. CancerResearch 50:1550-1558 (1990). In another example, cross-blocking studiescan be done with direct fluorescence on microtiter plates. In thismethod, the monoclonal antibody of interest is conjugated withfluorescein isothiocyanate (FITC), using established procedures (Wofsyet al. Selected Methods in Cellular Immunology, p. 287, Mishel andSchiigi (eds.) San Francisco: W.J. Freeman Co. (1980)). The selectedIFN-α is layered onto the wells of microtiter plates, the layered wellsare incubated with mixtures of (1) FITC-labeled monoclonal antibody ofinterest and (2) unlabeled test monoclonal antibody, and thefluorescence in each well is quantitated to determine the level ofcross-blocking exhibited by the antibodies. Monoclonal antibodies areconsidered to share an epitope if each blocks binding of the other by50% or greater in comparison to an irrelevant monoclonal antibodycontrol.

The results obtained in the cell-based biological assays can then befollowed by testing in animal, e.g. murine, models, and human clinicaltrials. If desired, murine monoclonal antibodies identified as havingthe desired properties can be converted into chimeric antibodies, orhumanized by techniques well known in the art, including the “geneconversion mutagenesis” strategy, as described in U.S. Pat. No.5,821,337, the entire disclosure of which is hereby expresslyincorporated by reference. Humanization of a particular anti-IFN-αantibody herein is also described in the Examples below.

(iv) Phage Display Method

Anti-IFN-α antibodies of the invention can be identified by usingcombinatorial libraries to screen for synthetic antibody clones with thedesired activity or activities. In principle, synthetic antibody clonesare selected by screening phage libraries containing phage that displayvarious fragments (e.g. Fab, F(ab′)₂, etc.) of antibody variable region(Fv) fused to phage coat protein. Such phage libraries are panned byaffinity chromatography against the desired antigen. Clones expressingFv fragments capable of binding to the desired antigen are adsorbed tothe antigen and thus separated from the non-binding clones in thelibrary. The binding clones are then eluted from the antigen, and can befurther enriched by additional cycles of antigen adsorption/elution. Anyof the anti-IFN-α antibodies of the invention can be obtained bydesigning a suitable antigen screening procedure to select for the phageclone of interest followed by construction of a full length anti-IFN-αantibody clone using the Fv sequences from the phage clone of interestand suitable constant region (Fc) sequences described in Kabat et al.(1991), supra.

Construction of Phage Display Libraries

The antigen-binding domain of an antibody is formed from two variable(V) regions of about 110 amino acids each, from the light (VL) and heavy(VH) chains, that both present three hypervariable loops orcomplementarity-determining regions (CDRs). Variable domains can bedisplayed functionally on phage, either as single-chain Fv (scFv)fragments, in which VH and VL are covalently linked through a short,flexible peptide, or as or D(ab′)₂ fragments, in which they are eachfused to a constant domain and interact non-covalently, as described inWinter et al., Ann. Rev. Immunol., 12: 433-455 (1994). As used herein,scFv encoding phage clones and Fab or F(ab′)₂ encoding phage clones arecollectively referred to as “Fv phage clones” or “Fv clones”.

The naive repertoire of an animal (the repertoire before antigenchallenge) provides it with antibodies that can bind with moderateaffinity (K_(d) ⁻¹ of about 10⁶ to 10⁷ M⁻¹) to essentially any non-selfmolecule. The sequence diversity of antibody binding sites is notencoded directly in the germline but is assembled in a combinatorialmanner from V gene segments. In human heavy chains, the first twohypervariable loops (H1 and H2) are drawn from less than 50 VH genesegments, which are combined with D segments and JH segments to createthe third hypervariable loop (H3). In human light chains, the first twohypervariable loops (L1 and L2) and much of the third (L3) are drawnfrom less than approximately 30 Vλ and less than approximately 30 Vκsegments to complete the third hypervariable loop (L3).

Each combinatorial rearrangement of V-gene segments in stem cells givesrise to a B cell that expresses a single VH-VL combination.Immunizations triggers any B cells making a combination that binds theimmunogen to proliferate (clonal expansion) and to secrete thecorresponding antibody. These naive antibodies are then matured to highaffinity (K_(d) ⁻¹ of 10⁹-10¹⁰ M⁻¹) by a process of mutagenesis andselection known as affinity maturation. It is after this point thatcells are normally removed to prepare hybridomas and generatehigh-affinity monoclonal antibodies.

At three stages of this process, repertoires of VH and VL genes can beseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be searched forantigen-binding clones as described in Winter et al., Ann. Rev.Immunol., 12: 433-455 (1994). Libraries from immunized sources providehigh-affinity antibodies to the immunogen without the requirement ofconstructing hybridomas. Alternatively, the naive repertoire can becloned to provide a single source of human antibodies to a wide range ofnon-self and also self antigens without any immunization as described byGriffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive librariescan also be made synthetically by cloning the unrearranged V- orVIE-gene segments from stem cells, and using PCR primers containingrandom sequence to encode the highly variable CDR3 regions and toaccomplish rearrangement in vitro as described by Hoogenboom and Winter,J. Mol. Biol., 227: 381-388 (1992).

Phage display mimics the B cell. Filamentous phage is used to displayantibody fragments by fusion to the minor coat protein pIII. Theantibody fragments can be displayed as single chain Fv fragments, inwhich VH and VL domains are connected on the same polypeptide chain by aflexible polypeptide spacer, e.g. as described by Marks et al., J. Mol.Biol., 222: 581-597 (1991), or as Fab (including F(ab′)₂) fragments, inwhich one chain is fused to pIII and the other is secreted into thebacterial host cell periplasm where assembly of a Fab-coat proteinstructure which becomes displayed on the phage surface by displacingsome of the wild type coat proteins, e.g. as described in Hoogenboom etal., Nucl. Acids Res., 19: 4133-4137 (1991). When antibody fragments arefused to the N-terminus of pIII, the phage is infective. However, if theN-terminal domain of pIII is excised and fusions made to the seconddomain, the phage is not infective, and wild type pIII must be providedby helper phage.

The pIII fusion and other proteins of the phage can be encoded entirelywithin the same phage replicon, or on different replicons. When tworeplicons are used, the pIII fusion is encoded on a phagemid, a plasmidcontaining a phage origin of replication. Phagemids can be packaged intophage particles by “rescue” with a helper phage such as M13K07 thatprovides all the phage proteins, including pIII, but due to a defectiveorigin is itself poorly packaged in competitions with the phagemids asdescribed in Vieira and Messing, Meth. Enzymol., 153: 3-11 (1987). In apreferred method, the phage display system is designed such that therecombinant phage can be grown in host cells under conditions permittingno more than a minor amount of phage particles to display more than onecopy of the Fv-coat protein fusion on the surface of the particle asdescribed in Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690(PCT/US91/09133 published Jun. 11, 1992).

In general, nucleic acids encoding antibody gene fragments are obtainedfrom immune cells harvested from humans or animals. If a library biasedin favor of anti-IFN-α clones is desired, the subject is immunized withIFN-α to generate an antibody response, and spleen cells and/orcirculating B cells other peripheral blood lymphocytes (PBLs) arerecovered for library construction. In a preferred embodiment, a humanantibody gene fragment library biased in favor of anti-human IFN-αclones is obtained by generating an anti-human IFN-α antibody responsein transgenic mice carrying a functional human immunoglobulin gene array(and lacking a functional endogenous antibody production system) suchthat IFN-α immunization gives rise to B cells producing human-sequenceantibodies against IFN-α.

In another preferred embodiment, animals are immunized with a mixture ofvarious, preferably all, IFN-α subtypes in order to generate an antibodyresponse that includes B cells producing anti-IFN-α antibodies withbroad reactivity against IFN-α subtypes. In another preferredembodiment, animals are immunized with the mixture of human IFN-αsubtypes that is present in the human lymphoblastoid interferonssecreted by Burkitt lymphoma cells (Namalva cells) induced with Sendaivirus, as described in Example 1 below. A suitable preparation of suchhuman lymphoblastoid interferons can be obtained commercially (ProductNo. 1-9887) from Sigma Chemical Company, St. Louis, Mo.

Additional enrichment for anti-IFN-α reactive cell populations can beobtained by using a suitable screening procedure to isolate B cellsexpressing IFN-α-specific membrane bound antibody, e.g., by cellseparation with IFN-α affinity chromatography or adsorption of cells tofluorochrome-labeled IFN-α followed by fluorescence-activated cellsorting (FACS).

Alternatively, the use of spleen cells and/or B cells or other PBLs froman unimmunized donor provides a better representation of the possibleantibody repertoire, and also permits the construction of an antibodylibrary using any animal (human or non-human) species in which IFN-α isnot antigenic. For libraries incorporating in vitro antibody geneconstruction, stem cells are harvested from the subject to providenucleic acids encoding unrearranged antibody gene segments. The immunecells of interest can be obtained from a variety of animal species, suchas human, mouse, rat, lagomorpha, luprine, canine, feline, porcine,bovine, equine, and avian species, etc.

Nucleic acid encoding antibody variable gene segments (including VH andVL segments) are recovered from the cells of interest and amplified. Inthe case of rearranged VH and VL gene libraries, the desired DNA can beobtained by isolating genomic DNA or mRNA from lymphocytes followed bypolymerase chain reaction (PCR) with primers matching the 5′ and 3′ endsof rearranged VH and VL genes as described in Orlandi et al., Proc.Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse Vgene repertoires for expression. The V genes can be amplified from cDNAand genomic DNA, with back primers at the 5′ end of the exon encodingthe mature V-domain and forward primers based within the J-segment asdescribed in Orlandi et al. (1989) and in Ward et al., Nature, 341:544-546 (1989). However, for amplifying from cDNA, back primers can alsobe based in the leader exon as described in Jones et al., Biotechnol.,9: 88-89 (1991), and forward primers within the constant region asdescribed in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732(1989). To maximize complementarity, degeneracy can be incorporated inthe primers as described in Orlandi et al. (1989) or Sastry et al.(1989). Preferably, the library diversity is maximized by using PCRprimers targeted to each V-gene family in order to amplify all availableVH and VL arrangements present in the immune cell nucleic acid sample,e.g. as described in the method of Marks et al., J. Mol. Biol., 222:581-597 (1991) or as described in the method of Orum et al., NucleicAcids Res., 21: 4491-4498 (1993). For cloning of the amplified DNA intoexpression vectors, rare restriction sites can be introduced within thePCR primer as a tag at one end as described in Orlandi et al. (1989), orby further PCR amplification with a tagged primer as described inClackson et al., Nature, 352: 624-628 (1991).

Repertoires of synthetically rearranged V genes can be derived in vitrofrom V gene segments. Most of the human VH-gene segments have beencloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet.,3: 88-94 (1993); these cloned segments (including all the majorconformations of the H1 and H2 loop) can be used to generate diverse VHgene repertoires with PCR primers encoding H3 loops of diverse sequenceand length as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be made with all the sequencediversity focussed in a long H3 loop of a single length as described inBarbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). HumanVκ and Vλ segments have been cloned and sequenced (reported in Williamsand Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used tomake synthetic light chain repertoires. Synthetic V gene repertoires,based on a range of VH and VL folds, and L3 and H3 lengths, will encodeantibodies of considerable structural diversity. Following amplificationof V-gene encoding DNAs, germline V-gene segments can be rearranged invitro according to the methods of Hoogenboom and Winter, J. Mol. Biol.,227: 381-388 (1992).

Repertoires of antibody fragments can be constructed by combining VH andVL gene repertoires together in several ways. Each repertoire can becreated in different vectors, and the vectors recombined in vitro, e.g.,as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo bycombinatorial infection, e.g., the loxP system described in Waterhouseet al., Nucl. Acids Res., 21: 2265-2266 (1993). The in vivorecombination approach exploits the two-chain nature of Fab fragments toovercome the limit on library size imposed by E. coli transformationefficiency. Naive VH and VL repertoires are cloned separately, one intoa phagemid and the other into a phage vector. The two libraries are thencombined by phage infection of phagemid-containing bacteria so that eachcell contains a different combination and the library size is limitedonly by the number of cells present (about 10¹² clones). Both vectorscontain in vivo recombination signals so that the VH and VL genes arerecombined onto a single replicon and are co-packaged into phagevirions. These huge libraries provide large numbers of diverseantibodies of good affinity (K_(d) ⁻¹ of about 10⁻⁸ M).

Alternatively, the repertoires may be cloned sequentially into the samevector, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA,88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g.as described in Clackson et al., Nature, 352: 624-628 (1991). PCRassembly can also be used to join VH and VL DNAs with DNA encoding aflexible peptide spacer to form single chain Fv (scFv) repertoires. Inyet another technique, “in cell PCR assembly” is used to combine VH andVL genes within lymphocytes by PCR and then clone repertoires of linkedgenes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837(1992).

The antibodies produced by naive libraries (either natural or synthetic)can be of moderate affinity (K_(d) ⁻¹ of about 10⁶ to 10⁷ M⁻¹), butaffinity maturation can also be mimicked in vitro by constructing andreselecting from secondary libraries as described in Winter et al.(1994), supra. For example, mutation can be introduced at random invitro by using error-prone polymerase (reported in Leung et al.,Technique, 1: 11-15 (1989)) in the method of Hawkins et al., J. Mol.Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl.Acad. Sci. USA, 89: 3576-3580 (1992). Additionally, affinity maturationcan be performed by randomly mutating one or more CDRs, e.g. using PCRwith primers carrying random sequence spanning the CDR of interest, inselected individual Fv clones and screening for higher affinity clones.WO 9607754 (published 14 Mar. 1996) described a method for inducingmutagenesis in a complementarity determining region of an immunoglobulinlight chain to create a library of light chain genes. Another effectiveapproach is to recombine the VH or VL domains selected by phage displaywith repertoires of naturally occurring V domain variants obtained fromunimmunized donors and screen for higher affinity in several rounds ofchain reshuffling as described in Marks et al., Biotechnol., 10: 779-783(1992). This technique allows the production of antibodies and antibodyfragments with affinities in the 10⁻⁹ M range.

Panning Phage Display Libraries for Anti-IFN-α Clones

a. Synthesis of IFN-α

Nucleic acid sequence encoding the IFN-α subtypes used herein can bedesigned using published amino acid and nucleic acid sequences ofinterferons, e.g. see the J. Interferon Res., 13: 443-444 (1993)compilation of references containing genomic and cDNA sequences forvarious type I interferons, and the references cited therein. For theIFN-αA (IFN-α2), IFN-αB (IFN-α8), IFN-αC (IFN-α10), IFN-αD (IFN-α1),IFN-αE (IFN-α22), IFN-αF (IFN-α21), IFN-αG (IFN-α5), and IFN-αH(IFN-α14) amino acid sequences or cDNA sequences, see FIGS. 3 and 4 onpages 23-24 of Goeddel et al., Nature, 290: 20-26 (1981). For cDNAencoding the amino acid sequence of IFN-α7 (IFN-αJ), see Cohen et al.,Dev. Biol. Standard, 60: 111-122 (1985). DNAs encoding the interferonsof interest can be prepared by a variety of methods known in the art.These methods include, but are not limited to, chemical synthesis by anyof the methods described in Engels et al., Agnew. Chem. Int. Ed. Engl.,28: 716-734 (1989), such as the triester, phosphite, phosphoramidite andH-phosphonate methods. In one embodiment, codons preferred by theexpression host cell are used in the design of the interferon-encodingDNA. Alternatively, DNA encoding the interferon can be isolated from agenomic or cDNA library.

Following construction of the DNA molecule encoding the interferon ofinterest, the DNA molecule is operably linked to an expression controlsequence in an expression vector, such as a plasmid, wherein the controlsequence is recognized by a host cell transformed with the vector. Ingeneral, plasmid vectors contain replication and control sequences whichare derived from species compatible with the host cell. The vectorordinarily carries a replication site, as well as sequences which encodeproteins that are capable of providing phenotypic selection intransformed cells.

For expression in prokaryotic hosts, suitable vectors include pBR322(ATCC No. 37,017), phGH107 (ATCC No. 40,011), pBO475, pS0132, pRIT5, anyvector in the pRIT20 or pRIT30 series (Nilsson and Abrahmsen, Meth.Enzymol., 185: 144-161 (1990)), pRIT2T, pKK233-2, pDR540 and pPL-lambda.Prokaryotic host cells containing the expression vectors suitable foruse herein include E. coli K12 strain 294 (ATCC NO. 31446), E colistrain JM101 (Messing et al., Nucl. Acid Res., 9: 309 (1981)), E. colistrain B, E. coli strain χ1776 (ATCC No. 31537), E. coli c600(Appleyard, Genetics, 39: 440 (1954)), E. coli W3110 (F-, gamma-,prototrophic, ATCC No. 27325), E. coli strain 27C7 (W3110, tonA, phoAE15, (argF-lac)169, ptr3, degP41, ompT, kan^(r)) (U.S. Pat. No.5,288,931, ATCC No. 55,244), Bacillus subtilis, Salmonella typhimurium,Serratia marcesans, and Pseudomonas species.

In addition to prokaryotes, eukaryotic organisms, such as yeasts, orcells derived from multicellular organisms can be used as host cells.For expression in yeast host cells, such as common baker's yeast orSaccharomyces cerevisiae, suitable vectors include episomallyreplicating vectors based on the 2-micron plasmid, integration vectors,and yeast artificial chromosome (YAC) vectors. For expression in insecthost cells, such as Sf9 cells, suitable vectors include baculoviralvectors. For expression in plant host cells, particularly dicotyledonousplant hosts, such as tobacco, suitable expression vectors includevectors derived from the Ti plasmid of Agrobacterium tumefaciens.

However, interest has been greatest in vertebrate host cells. Examplesof useful mammalian host cells include monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol., 36: 59 (1977)); baby hamster kidney cells (BHK, ATCCCCL 10); Chinese hamster ovary cells/-DHFR(CHO, Urlaub and Chasin, Proc.Natl. Acad. Sci. USA, 77: 4216 (1980)); mouse sertoli cells (TM4,Mather, Biol. Reprod., 23: 243-251 (1980)); monkey kidney cells (CV1ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCCCRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); caninekidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCCCRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells(Mather et al., Annals N.Y. Acad. Sci., 383: 44-68 (1982)); MRC 5 cells;FS4 cells; and a human hepatoma cell line (Hep G2). For expression inmammalian host cells, useful vectors include vectors derived from SV40,vectors derived from cytomegalovirus such as the pRK vectors, includingpRK5 and pRK7 (Suva et al., Science, 237: 893-896 (1987), EP 307,247(Mar. 15, 1989), EP 278,776 (Aug. 17, 1988)) vectors derived fromvaccinia viruses or other pox viruses, and retroviral vectors such asvectors derived from Moloney's murine leukemia virus (MoMLV).

Optionally, the DNA encoding the interferon of interest is operablylinked to a secretory leader sequence resulting in secretion of theexpression product by the host cell into the culture medium. Examples ofsecretory leader sequences include stil, ecotin, lamB, herpes GD, lpp,alkaline phosphatase, invertase, and alpha factor. Also suitable for useherein is the 36 amino acid leader sequence of protein A (Abrahmsen etal., EMBO J., 4: 3901 (1985)).

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors of this invention andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ precipitation and electroporation. Successfultransfection is generally recognized when any indication of theoperation of this vector occurs within the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in section 1.82 of Sambrook etal., Molecular Cloning (2nd ed.), Cold Spring Harbor Laboratory, NY(1989), is generally used for prokaryotes or other cells that containsubstantial cell-wall barriers. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23: 315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method described in sections 16.30-16.37 of Sambrook etal., supra, is preferred. General aspects of mammalian cell host systemtransformations have been described by Axel in U.S. Pat. No. 4,399,216issued 16 Aug. 1983. Transformations into yeast are typically carriedout according to the method of Van Solingen et al., J. Bact., 130: 946(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76: 3829 (1979).However, other methods for introducing DNA into cells such as by nuclearinjection, electroporation, or by protoplast fusion may also be used.

Prokaryotic host cells used to produce the interferon of interest can becultured as described generally in Sambrook et al., supra.

The mammalian host cells used to produce the interferon of interest canbe cultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham and Wallace, Meth. Enz., 58: 44 (1979), Barnes andSato, Anal. Biochem., 102: 255 (1980), U.S. Pat. No. 4,767,704;4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195; U.S. Pat.Re. 30,985; or U.S. Pat. No. 5,122,469, the disclosures of all of whichare incorporated herein by reference, may be used as culture media forthe host cells. Any of these media may be supplemented as necessary withhormones and/or other growth factors (such as insulin, transferrin, orepidermal growth factor), salts (such as sodium chloride, calcium,magnesium, and phosphate), buffers (such as HEPES), nucleosides (such asadenosine and thymidine), antibiotics (such as Gentamycin™ drug), traceelements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range), and glucose or an equivalentenergy source. Any other necessary supplements may also be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, pH, and the like, arethose previously used with the host cell selected for expression, andwill be apparent to the ordinarily skilled artisan.

The host cells referred to in this disclosure encompass cells in invitro culture as well as cells that are within a host animal.

In an intracellular expression system or periplasmic space secretionsystem, the recombinantly expressed interferon protein can be recoveredfrom the culture cells by disrupting the host cell membrane/cell wall(e.g. by osmotic shock or solubilizing the host cell membrane indetergent). Alternatively, in an extracellular secretion system, therecombinant protein can be recovered from the culture medium. As a firststep, the culture medium or lysate is centrifuged to remove anyparticulate cell debris. The membrane and soluble protein fractions arethen separated. Usually, the interferon is purified from the solubleprotein fraction. If the IFN-α is expressed as a membrane bound species,the membrane bound peptide can be recovered from the membrane fractionby solubilization with detergents. The crude peptide extract can then befurther purified by suitable procedures such as fractionation onimmunoaffinity or ion-exchange columns; ethanol precipitation; reversephase HPLC; chromatography on silica or on a cation exchange resin suchas DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; hydrophobic affinityresins and ligand affinity using interferon receptor immobilized on amatrix.

Many of the human IFN-α used herein can be obtained from commercialsources, e.g. from Sigma (St. Louis, Mo.), Calbiochem-NovabiochemCorporation (San Diego, Calif.) or ACCURATE Chemical & ScientificCorporation (Westbury, N.Y.).

Standard cloning procedures described in Maniatis et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989) are used to construct plasmids that directthe translocation of the various species of hIFN-α into the periplasmicspace of E. coli. PCR reactions are performed on cDNA clones of thevarious subspecies of hIFN-α disclosed in Goeddel et al., Nature 290:20-26 (1981) with NsiI and StyI restriction sites added to the primers.These PCR products are then subcloned into the corresponding sites ofthe expression vector pB0720 described in Cunningham et al., Science243:1330-1336 (1989). The resulting plasmids place production of thehIFN-α subtypes under control of the E. coli phoA promoter and theheat-stable enterotoxin II signal peptide as described in Chang et al.,Gene 55: 189-196 (1987). The correct DNA sequence of each gene isconfirmed using the United States Biochemical Sequenase Kit version 2.0.Each plasmid is transformed into the E. coli strain 27C7 (ATCC #55244)and grown in 10 liter fermentors as described in Carter et al.,Bio/Technology 10: 163-167 (1992). Human hIFNs are purified from E. colipaste containing each IFN-α by affinity chromatography. Bacterial cellsare lysed, and the lysate is centrifuged at 10,000×g to remove debris.The supernatant is applied to an immunoaffinity column containing amouse anti-hIFN-αB antibody (LI-1) that is obtained as described inStaehelin et al., Proc. Natl. Acad. Sci. 78:1848-1852 (1981). LI-1 isimmobilized on controlled pore glass by a modification of the method ofRoy et al., Journal of Chromatography, 303: 225-228 (1984). The boundinterferon is eluted from the column with 0.1 M citrate, pH 3.0,containing 20% (w/v) glycerol. The purified IFN is analyzed by SDS-PAGEand immunoblotting, and is assayed for bioactivity by the hIFN-inducedanti-viral assay as described herein.

Human IFN-α2/1 hybrid molecule (IFN-α2₁₋₆₂/α₆₄₋₁₆₆) was obtained asdescribed in Rehberg et al., J. Biol. Chem., 257: 11497-11502 (1992) orHorisberger and Marco, Pharmac. Ther., 66: 507-534 (1995).

b. Immobilization of IFN-α

The purified IFN-α can be attached to a suitable matrix such as agarosebeads, acrylamide beads, glass beads, cellulose, various acryliccopolymers, hydroxyl methacrylate gels, polyacrylic and polymethacryliccopolymers, nylon, neutral and ionic carriers, and the like, for use inthe affinity chromatographic separation of phage display clones.Attachment of the IFN-α protein to the matrix can be accomplished by themethods described in Methods in Enzymology, vol. 44 (1976). A commonlyemployed technique for attaching protein ligands to polysaccharidematrices, e.g. agarose, dextran or cellulose, involves activation of thecarrier with cyanogen halides and subsequent coupling of the peptideligand's primary aliphatic or aromatic amines to the activated matrix.

Alternatively, IFN-α can be used to coat the wells of adsorption plates,expressed on host cells affixed to adsorption plates or used in cellsorting, or conjugated to biotin for capture with streptavidin-coatedbeads, or used in any other art-known method for panning phage displaylibraries.

c. Panning Procedures

The phage library samples are contacted with immobilized IFN-α underconditions suitable for binding of at least a portion of the phageparticles with the adsorbent. Normally, the conditions, including pH,ionic strength, temperature and the like are selected to mimicphysiological conditions. The phage bound to the solid phase are washedand then eluted by acid, e.g. as described in Barbas et al., Proc. Natl.Acad. Sci. USA, 88: 7978-7982 (1991), or by alkali, e.g. as described inMarks et al., J. Mol. Biol., 222: 581-597 (1991), or by IFN-α antigen,e.g. in a procedure similar to the antigen competition method ofClackson et al., Nature, 352: 624-628 (1991). Phage can be enriched20-1,000-fold in a single round of selection. Moreover, the enrichedphage can be grown in bacterial culture and subjected to further roundsof selection.

In a preferred embodiment, phage are serially incubated with variousIFN-α subtypes immobilized in order to identify and further characterizephage clones that exhibit appreciable binding to a majority, preferablyall, of IFN-α subtypes. In this method, phage are first incubated withone specific IFN-α subtype. The phage bound to this subtype are elutedand subjected to selection with another IFN-α subtype. The process ofbinding and elution is thus repeated with all IFN-α subtypes. At theend, the procedure yields a population of phage displaying antibodieswith broad reactivity against all IFN-α subtypes. These phage can thenbe tested against other IFN species, i.e. other than IFN-α, in order toselect those clones which do not show appreciable binding to otherspecies of IFNs. Finally, the selected phage clones can be examined fortheir ability to neutralize biological activity, e.g. anti-viralactivity, of various IFN-α subtypes, and clones representing antibodieswith broad neutralization activity against a majority, preferably all,of IFN-α subtypes are finally selected.

The efficiency of selection depends on many factors, including thekinetics of dissociation during washing, and whether multiple antibodyfragments on a single phage can simultaneously engage with antigen.Antibodies with fast dissociation kinetics (and weak binding affinities)can be retained by use of short washes, multivalent phage display andhigh coating density of antigen in solid phase. The high density notonly stabilizes the phage through multivalent interactions, but favorsrebinding of phage that has dissociated. The selection of antibodieswith slow dissociation kinetics (and good binding affinities) can bepromoted by use of long washes and monovalent phage display as describedin Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and alow coating density of antigen as described in Marks et al.,Biotechnol., 10: 779-783 (1992).

It is possible to select between phage antibodies of differentaffinities, even with affinities that differ slightly, for IFN-α.However, random mutation of a selected antibody (e.g. as performed insome of the affinity maturation techniques described above) is likely togive rise to many mutants, most binding to antigen, and a few withhigher affinity. With limiting IFN-α, rare high affinity phage could becompeted out. To retain all the higher affinity mutants, phage can beincubated with excess biotinylated IFN-α, but with the biotinylatedIFN-α at a concentration of lower molarity than the target molaraffinity constant for IFN-α. The high affinity-binding phage can then becaptured by streptavidin-coated paramagnetic beads. Such “equilibriumcapture” allows the antibodies to be selected according to theiraffinities of binding, with sensitivity that permits isolation of mutantclones with as little as two-fold higher affinity from a great excess ofphage with lower affinity. Conditions used in washing phage bound to asolid phase can also be manipulated to discriminate on the basis ofdissociation kinetics.

In one embodiment, phage are serially incubated with various IFN-αsubtypes immobilized on a solid support, such as chromatographic polymermatrix beads described above. In this method, the phage are firstincubated with one specific IFN-α subtype. The phage bound to thissubtype are eluted from solid phase with a suitable eluent, such as anysalt or acid buffer capable of releasing the bound phage into solution.Next, the eluted phage clones are subjected to selection with anotherIFN-α subtype. In order to enrich the population for clones that competewith soluble IFNAR2 for binding to IFN-α, the phage clones recoveredfrom the series of IFN-α subtype chromatographic separations areincubated with a complex of immobilized IFNAR2 preadsorbed to IFN-α, andthe non-adsorbed phage clones are recovered from the incubation reactionmixture.

The selection procedures can be designed to utilize any suitable batchchromatographic technique. In one embodiment, the phage clones areadsorbed to IFN-α-derivatized polymer matrix beads in suspension, theadsorbed beads are recovered by centrifugation, the recovered beads areresuspended and incubated in a suitable elution buffer, such as any saltor acid buffer capable of releasing the bound phage into solution, theelution mixture is centrifuged, the eluted phage clones are recoveredfrom the supernatant, and then the adsorption/elution procedure isrepeated for every additional IFN-α subtype. In order to enrich thepopulation for clones that compete with soluble IFNAR2 for binding toIFN-α, the phage clones recovered from the IFN-α subtype chromatographicseparations are incubated with a suspension of IFNAR2-derivatizedpolymer matrix beads preadsorbed to IFN-α, the incubation mixture iscentrifuged, and the non-adsorbed phage clones are recovered from thesupernatant.

In another embodiment, the selection procedure is designed to enrich thephage population for the property of inhibiting IFN-α binding to IFNAR2during each of the affinity chromatographic separations. In this method,the phage are serially incubated with each of the specific IFN-αsubtypes immobilized on a solid support and then eluted from solid phasewith an eluent comprising an excess of soluble IFNAR2, such asIFNAR2ECD-IgG Fc, under conditions wherein soluble IFNAR2 is capable ofdisplacing any phage clone that competes with IFNAR2 for binding to theimmobilized IFN-α. The process of binding and elution is thus repeatedwith each of the specific IFN-α subtypes.

In another embodiment, the phage clones are adsorbed toIFN-α-derivatized polymer matrix beads in suspension, the adsorbed beadsare recovered by centrifugation, the recovered beads are resuspended andincubated in a suitable elution buffer comprising an excess of solubleIFNAR2 (such as IFNAR2ECD-IgG Fc) under conditions wherein the solubleIFNAR2 is capable of displacing any phage clone that competes withIFNAR2 for binding to the immobilized IFN-α and releasing the boundphage into solution, the elution mixture is centrifuged, the elutedphage clones are recovered from the supernatant, and then theadsorption/elution procedure is repeated for every additional IFN-αsubtype.

At the end, the procedure yields a population of phage displayingantibodies with IFNAR2-binding inhibition activity against a broad rangeof IFN-α subtypes. These phage can then be tested against other IFNspecies (other than IFN-α species), such as IFN-β, in order to selectthose clones which do not show appreciable binding to other species ofIFNs. Finally, the selected phage clones can be examined for theirability to neutralize biological activity, e.g. anti-viral activity, ofvarious IFN-α subtypes, and clones representing antibodies with broadneutralization activity against a majority, preferably all, of IFN-αsubtypes are finally selected.

Activity Selection of Anti-IFN-α Clones

In one embodiment, the invention provides anti-IFN-α antibodies thatbind to as well as neutralize the activity of a majority, preferablyall, of IFN-α subtypes, but do not significantly bind to or neutralizethe activity of any other interferon species. For example, the abilityof various phage clones to neutralize the anti-viral activities ofvarious IFN-α subtypes can be tested, essentially in the same manner asdescribed earlier for the antibodies.

4. Preparation of Soluble IFNAR2-IgG

A cDNA encoding the human immunoglobulin fusion proteins(immunoadhesins) based on the extracellular domain (ECD) of the hIFNAR2(pRK5 hIFNAR2-IgG clone) can be generated using methods similar to thosedescribed by Haak-Frendscho et al., Immunology 79: 594-599 (1993) forthe construction of a murine IFN-γ receptor immunoadhesin. Briefly, theplasmid pRKCD4₂Fc₁ is constructed as described in Example 4 of WO89/02922 (PCT/US88/03414 published Apr. 6, 1989). The cDNA codingsequence for the first 216 residues of the mature hIFNAR2ECD is obtainedfrom the published sequence (Novick et al., Cell, 77: 391-400 [1994]).The CD4 coding sequence in the pRKCD4₂Fc₁ is replaced with thehIFNAR2ECD encoding cDNA to form the pRK5hIFNAR2-IgG clone. hIFNAR2-IgGis expressed in human embryonic kidney 293 cells by transienttransfection using a calcium phosphate precipitation technique. Theimmunoadhesin is purified from serum-free cell culture supernatants in asingle step by affinity chromatography on a protein A-sepharose columnas described in Haak-Frendscho et al. (1993), supra. Bound hIFNAR2-IgGis eluted with 0.1 M citrate buffer, pH 3.0, containing 20% (w/v)glycerol. The hIFNAR2-IgG purified is over 95% pure, as judged bySDS-PAGE.

5. Diagnostic Uses of Anti-IFN-α Antibodies

The anti-IFN-α antibodies of the invention are unique research reagentsin diagnostic assays for IFN-α expression. As discussed earlier, IFN-αexpression is increased in certain autoimmune diseases such as IDDM,SLE, and autoimmune thyroiditis. Increased expression of various IFN-αsubtypes in such disorders can be detected and quantitated usinganti-IFN-α antibodies of the instant invention with broad reactivityagainst a majority of IFN-α subtypes. Anti-IFN-α antibodies are alsouseful for the affinity purification of various IFN-α subtypes fromrecombinant cell culture or natural sources.

Anti-IFN-α antibodies can be used for the detection of IFN-α in any oneof a number of well known diagnostic assay methods. For example, abiological sample may be assayed for IFN-α by obtaining the sample froma desired source, admixing the sample with anti-IFN-α antibody to allowthe antibody to form antibody/IFN-α complex with any IFN-α subtypepresent in the mixture, and detecting any antibody/IFN-α complex presentin the mixture. The biological sample may be prepared for assay bymethods known in the art which are suitable for the particular sample.The methods of admixing the sample with antibodies and the methods ofdetecting antibody/IFN-α complex are chosen according to the type ofassay used. Such assays include competitive and sandwich assays, andsteric inhibition assays. Competitive and sandwich methods employ aphase-separation step as an integral part of the method while stericinhibition assays are conducted in a single reaction mixture.

Analytical methods for IFN-α all use one or more of the followingreagents: labeled IFN-α analogue, immobilized IFN-α analogue, labeledanti-IFN-α antibody, immobilized anti-IFN-α antibody and stericconjugates. The labeled reagents also are known as “tracers.”

The label used is any detectable functionality that does not interferewith the binding of IFN-α and anti-IFN-α antibody. Numerous labels areknown for use in immunoassay, examples including moieties that may bedetected directly, such as fluorochrome, chemiluminescent, andradioactive labels, as well as moieties, such as enzymes, that must bereacted or derivatized to be detected. Examples of such labels includethe radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I, fluorophores such asrare earth chelates or fluorescein and its derivatives, rhodamine andits derivatives, dansyl, umbelliferone, luceriferases, e.g., fireflyluciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,saccharide oxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

Conventional methods are available to bind these labels covalently toproteins or polypeptides. For instance, coupling agents such asdialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotizedbenzidine, and the like may be used to tag the antibodies with theabove-described fluorescent, chemiluminescent, and enzyme labels. See,for example, U.S. Pat. Nos. 3,940,475 (fluorimetry) and 3,645,090(enzymes); Hunter et al., Nature, 144: 945 (1962); David et al.,Biochemistry, 13: 1014-1021 (1974); Pain et al., J. Immunol. Methods,40: 219-230 (1981); and Nygren, J. Histochem. and Cytochem., 30: 407-412(1982). Preferred labels herein are enzymes such as horseradishperoxidase and alkaline phosphatase.

The conjugation of such label, including the enzymes, to the antibody isa standard manipulative procedure for one of ordinary skill inimmunoassay techniques. See, for example, O'Sullivan et al., “Methodsfor the Preparation of Enzyme-antibody Conjugates for Use in EnzymeImmunoassay,” in Methods in Enzymology, ed. J. J. Langone and H. VanVunakis, Vol. 73 (Academic Press, New York, N.Y., 1981), pp. 147-166.

Immobilization of reagents is required for certain assay methods.Immobilization entails separating the anti-IFN-α antibody from any IFN-αthat remains free in solution. This conventionally is accomplished byeither insolubilizing the anti-IFN-α antibody or IFN-α analogue beforethe assay procedure, as by adsorption to a water-insoluble matrix orsurface (Bennich et al., U.S. Pat. No. 3,720,760), by covalent coupling(for example, using glutaraldehyde cross-linking), or by insolubilizingthe anti-IFN-α antibody or IFN-α analogue afterward, e.g., byimmunoprecipitation.

Other assay methods, known as competitive or sandwich assays, are wellestablished and widely used in the commercial diagnostics industry.

Competitive assays rely on the ability of a tracer IFN-α analogue tocompete with the test sample IFN-α for a limited number of anti-IFN-αantibody antigen-binding sites. The anti-IFN-α antibody generally isinsolubilized before or after the competition and then the tracer andIFN-α bound to the anti-IFN-α antibody are separated from the unboundtracer and IFN-α. This separation is accomplished by decanting (wherethe binding partner was pre-insolubilized) or by centrifuging (where thebinding partner was precipitated after the competitive reaction). Theamount of test sample IFN-α is inversely proportional to the amount ofbound tracer as measured by the amount of marker substance.Dose-response curves with known amounts of IFN-α are prepared andcompared with the test results to quantitatively determine the amount ofIFN-α present in the test sample. These assays are called ELISA systemswhen enzymes are used as the detectable markers.

Another species of competitive assay, called a “homogeneous” assay, doesnot require a phase separation. Here, a conjugate of an enzyme with theIFN-α is prepared and used such that when anti-IFN-α antibody binds tothe IFN-α the presence of the anti-IFN-α antibody modifies the enzymeactivity. In this case, the IFN-α or its immunologically activefragments are conjugated with a bifunctional organic bridge to an enzymesuch as peroxidase. Conjugates are selected for use with anti-IFN-αantibody so that binding of the anti-IFN-α antibody inhibits orpotentiates the enzyme activity of the label. This method per se iswidely practiced under the name of EMIT.

Steric conjugates are used in steric hindrance methods for homogeneousassay. These conjugates are synthesized by covalently linking alow-molecular-weight hapten to a small IFN-α fragment so that antibodyto hapten is substantially unable to bind the conjugate at the same timeas anti-IFN-α antibody. Under this assay procedure the IFN-α present inthe test sample will bind anti-IFN-α antibody, thereby allowinganti-hapten to bind the conjugate, resulting in a change in thecharacter of the conjugate hapten, e.g., a change in fluorescence whenthe hapten is a fluorophore.

Sandwich assays particularly are useful for the determination of IFN-αor anti-IFN-α antibodies. In sequential sandwich assays an immobilizedanti-IFN-α antibody is used to adsorb test sample IFN-α, the test sampleis removed as by washing, the bound IFN-α is used to adsorb a second,labeled anti-IFN-α antibody and bound material is then separated fromresidual tracer. The amount of bound tracer is directly proportional totest sample IFN-α. In “simultaneous” sandwich assays the test sample isnot separated before adding the labeled anti-IFN-α. A sequentialsandwich assay using an anti-IFN-α monoclonal antibody as one antibodyand a polyclonal anti-IFN-α antibody as the other is useful in testingsamples for IFN-α.

The foregoing are merely exemplary diagnostic assays for IFN-α. Othermethods now or hereafter developed that use anti-IFN-α antibody for thedetermination of IFN-α are included within the scope hereof, includingthe bioassays described above.

6. Therapeutic Compositions and Administration of Anti-IFN-α Antibodies

Therapeutic formulations of the anti-IFN-α antibodies of the inventionare prepared for storage by mixing antibody having the desired degree ofpurity with optional physiologically acceptable carriers, excipients, orstabilizers (Remington: The Science and Practice of Pharmacy, 19thEdition, Alfonso, R., ed, Mack Publishing Co. (Easton, Pa.: 1995)), inthe form of lyophilized cake or aqueous solutions. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid; lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, Pluronics or polyethylene glycol (PEG).

The anti-IFN-α antibody to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes, prior to or following lyophilization andreconstitution. The anti-IFN-α antibody ordinarily will be stored inlyophilized form or in solution.

Therapeutic anti-IFN-α antibody compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

The route of anti-IFN-α antibody administration is in accord with knownmethods, e.g. injection or infusion by intravenous, intraperitoneal,intracerebral, subcutaneous, intramuscular, intraocular, intraarterial,intracerebrospinal, or intralesional routes, or by sustained releasesystems as noted below. Preferably the antibody is given systemically.

Suitable examples of sustained-release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices include polyesters,hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymersof L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al.,Biopolymers, 22: 547-556 (1983)), poly (2-hydroxyethyl-methacrylate)(Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981) and Langer,Chem. Tech., 12: 98-105 (1982)), ethylene vinyl acetate (Langer et al.,supra) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).Sustained-release anti-IFNAR2 antibody compositions also includeliposomally entrapped antibody. Liposomes containing antibody areprepared by methods known per se: DE 3,218,121; Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl.Acad. Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046;EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Pat.Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomesare of the small (about 200-800 Angstroms) unilamelar type in which thelipid content is greater than about 30 mol. % cholesterol, the selectedproportion being adjusted for the optimal antibody therapy.

Anti-IFN-α antibody can also be administered by inhalation. Commerciallyavailable nebulizers for liquid formulations, including jet nebulizersand ultrasonic nebulizers are useful for administration. Liquidformulations can be directly nebulized and lyophilized powder can benebulized after reconstitution. Alternatively, anti-IFN-α antibody canbe aerosolized using a fluorocarbon formulation and a metered doseinhaler, or inhaled as a lyophilized and milled powder.

An “effective amount” of anti-IFN-α antibody to be employedtherapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, the type of anti-IFN-α antibodyemployed, and the condition of the patient. Accordingly, it will benecessary for the therapist to titer the dosage and modify the route ofadministration as required to obtain the optimal therapeutic effect.Typically, the clinician will administer the anti-IFN-α antibody until adosage is reached that achieves the desired effect. The progress of thistherapy is easily monitored by conventional assays.

The patients to be treated with the anti-IFN-α antibody of the inventioninclude preclinical patients or those with recent onset ofimmune-mediated disorders, and particularly autoimmune disorders.Patients are candidates for therapy in accord with this invention untilsuch point as no healthy tissue remains to be protected fromimmune-mediated destruction. For example, a patient suffering frominsulin-dependent diabetes mellitus (IDDM) can benefit from therapy withan anti-IFN-α antibody of the invention until the patient's pancreaticislet cells are no longer viable. It is desirable to administer ananti-IFN-α antibody as early as possible in the development of theimmune-mediated or autoimmune disorder, and to continue treatment for aslong as is necessary for the protection of healthy tissue fromdestruction by the patient's immune system. For example, the IDDMpatient is treated until insulin monitoring demonstrates adequate isletresponse and other indicia of islet necrosis diminish (e.g. reduction inanti-islet antibody titers), after which the patient can be withdrawnfrom anti-IFN-α antibody treatment for a trial period during whichinsulin response and the level of anti-islet antibodies are monitoredfor relapse.

In the treatment and prevention of an immune-mediated or autoimmunedisorder by an anti-IFN-α antibody, the antibody composition will beformulated, dosed, and administered in a fashion consistent with goodmedical practice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the antibody, the particular type ofantibody, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of antibody to be administered willbe governed by such considerations, and is the minimum amount necessaryto prevent, ameliorate, or treat the disorder, including treatingchronic autoimmune conditions and immunosuppression maintenance intransplant recipients. Such amount is preferably below the amount thatis toxic to the host or renders the host significantly more susceptibleto infections.

As a general proposition, the initial pharmaceutically effective amountof the antibody administered parenterally will be in the range of about0.1 to 50 mg/kg of patient body weight per day, with the typical initialrange of antibody used being 0.3 to 20 mg/kg/day, more preferably 0.3 to15 mg/kg/day. The desired dosage can be delivered by a single bolusadministration, by multiple bolus administrations, or by continuousinfusion administration of antibody, depending on the pattern ofpharmacokinetic decay that the practitioner wishes to achieve.

As noted above, however, these suggested amounts of antibody are subjectto a great deal of therapeutic discretion. The key factor in selectingan appropriate dose and scheduling is the result obtained, as indicatedabove.

The antibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the immune-mediated orautoimmune disorder in question. For example, in rheumatoid arthritis,the antibody may be given in conjunction with a glucocorticosteroid. Theeffective amount of such other agents depends on the amount ofanti-IFN-α antibody present in the formulation, the type of disorder ortreatment, and other factors discussed above. These are generally usedin the same dosages and with administration routes as used hereinbeforeor about from 1 to 99% of the heretofore employed dosages.

Further details of the invention can be found in the following example,which further defines the scope of the invention. All references citedthroughout the specification, and the references cited therein, arehereby expressly incorporated by reference in their entirety.

EXAMPLES

The following examples are offered by way of illustration and not by wayof limitation. The examples are provided so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the compounds, compositions, and methods of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to insure accuracywith respect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviation should be accounted for. Unlessindicated otherwise, parts are parts by weight, temperature is indegrees C., and pressure is at or near atmospheric. The disclosures ofall citations in the specification are expressly incorporated herein byreference.

Example 1 Generation and Characterization of a Broad Reactive MouseAnti-IFN-α Monoclonal Antibody Materials and Methods

A Murine Monoclonal Antibody with Broad Reactivity Against IFN-αSubtypes

A pan-IFN-α neutralizing antibody was developed by sequentiallyimmunizing mice with the mixture of human IFN-α subtypes, generating alarge number of candidate mAbs, and then screening for binding andactivity. In particular, Balb/c mice were immunized into each hindfootpad 9 times (at two week intervals) with 2.5 μg of lymphoblastoidhIFN-α (Product No. 1-9887 of Sigma, St. Louis, Mo.) resuspended inMPL-TDM (Ribi Immunochemical Research, Inc., Hamilton, Mont.). Threedays after the final boost, popliteal lymph node cells were fused withmurine myeloma cells P3X63Ag8.U.1 (ATCC CRL1597), using 35% polyethyleneglycol. Hybridomas were selected in HAT medium. Ten days after thefusion, hybridoma culture supernatants were first screened for mAbsbinding to the various species of hIFN-α in an ELISA. The selectedhybridoma culture supernatants were then tested for their ability toinhibit the anti-viral cytophathic effect of IFN on human lung carcinomacell line A549 cells as described below. As indicated in FIG. 1, threemAbs obtained from 1794 fusion wells were able to neutralize a diverseset of IFN-α subtypes. These three mAbs were subcloned and re-analyzed.

Neutralization of Antiviral Activity of IFN-α

The ability of a candidate antibody to neutralize the antiviral activityof IFN-α was assayed as described by Yousefi, S., et al., Am. J. Clin.Pathol., 83: 735-740 (1985). Briefly, the assay was performed usinghuman lung carcinoma A549 cells challenged with encephalomyocarditis(EMC) virus. Serial dilutions of mAbs were incubated with various unitsof type I interferons for one hour at 37° C. in a total volume of 100μl. These mixtures were then incubated with 5×10⁵ A549 cells in 100 μlof cell culture medium for 24 hours. Cells were then challenged with2×10⁵ pfu of EMC virus for an additional 24 hours. At the end of theincubation, cell viability was determined by visual microscopicexamination or crystal violet staining. The neutralizing antibody titer(EC50) was defined as the concentration of antibody which neutralizes50% of the anti-viral cytopathic effect by 100 units/ml of type I IFNs.The units of type I IFNs used in this study were determined using NIHreference recombinant human IFN-α2 as a standard. The specificactivities of the various type I IFNs tested were as follows: IFN-α2/-α1(IFN-α2 residues 1-62/-α1 residues 64-166) (2×10⁷ IU/mg), IFN-α1 (3×10⁷IU/mg), IFN-α2 (2×10⁷ IU/mg), IFN-α5 (8×10⁷ IU/mg), IFN-α8 (19×10⁷IU/mg), and IFN-α10 (1.5×10⁵ IU/mg). The leukocyte IFN tested was SigmaProduct No. 1-2396. The lymphoblastoid IFN tested was NIH referencestandard Ga23-901-532. The data shown in FIG. 3B was obtained using theabove-described assay format in experiments performed by AccessBiomedical (San Diego, Calif.) at the behest of applicant.

Electrophoretic Mobility Shift Assay

Most of the immediate actions of IFN have been linked to activation oflatent cytoplasmic signal transducers and activators of transcription(STAT) proteins to produce a multiprotein complex, interferon-stimulatedgene factor-3 (ISGF3), which induces transcription from target promoterinterferon-stimulated response element (ISRE). ISGF3 is composed ofthree protein subunits: STAT1, STAT2 and p48/ISGF3γ. The p48 proteinbelongs to the interferon regulatory factor (IRF) family, and is aDNA-binding protein that directly interacts with ISRE. Thus, monitoringISRE specific cellular DNA-binding complex in response to IFN treatmentprovides a simple, rapid and convenient method to assess the effect ofIFN on target cells. One of the convenient formats to carry out such ananalysis is electrophoretic mobility shift assay (EMSA), wherein theinduction of an ISRE-binding activity by IFN treatment results in theshift in the electrophoretic mobility of a radiolabeled double-strandedoligonucleotide probe corresponding to the consensus sequence of ISRE.

The assay was carried out essentially as described by Kurabayashi etal., Mol. Cell. Biol., 15: 6386 (1995). Briefly, 5 ng of a specificIFN-α subtype plus various concentrations (5-100 μg/ml) of anti-IFN-α:mAbs were incubated with 5×10⁵ HeLa cells in 200 μl of DMEM for 30minutes at 37° C. Cells were preincubated with antibody for 15 minutesat 4° C. before the addition of the hIFN-α. Cells were washed in PBS andresuspended in 125 μl buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mMETDA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin,10 μg/ml aprotinin). After a 15 minute incubation on ice, cells werelysed by the addition of 0.025% NP40. The nuclear pellet was obtained bycentrifugation and was resuspended in 50 μl buffer B (20 mM HEPES, pH7.9, 400 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonylfluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin) and kept on ice for 30min. The nuclear fraction was cleared by centrifugation and thesupernatant stored at −70° C. until use. Double-stranded probes wereprepared from single-stranded oligonucleotides (ISG15 top:5′-GATCGGGAAAGGGAAACCGAAACTGAAGCC-3′ [SEQ ID NO. 13], ISG15 bottom:5′-GATCGGCTTCAGTTTCGGTTTCCCTTTCCC-3′ [SEQ ID NO. 14]) utilizing a DNApolymerase I Klenow filling reaction with ³²P-dATP (3,000 Ci/mM,Amersham). Labeled oligonucleotides were purified from unincorporatedradioactive nucleotides using BIO-Spin 30 columns (Bio-Rad). Bindingreactions containing 5 μl nuclear extract, 25,000 cpm of labeled probeand 2 μg of non-specific competitor poly (dI-dC)-poly (dI-dC) in 15 μlbinding buffer (10 mM Tris-HCL, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT,1 mM phenylmethylsulfonyl fluoride and 15% glycerol) were incubated atRT for 30 minutes. DNA-protein complexes were resolved in 6%non-denaturing polyacrylamide gels and analyzed by autoradiograph. Thespecificity of the assay was determined by the addition of 350 ng ofunlabeled ISG15 probe in separate reaction mixtures. Formation of anISGF3 specific complex was confirmed by a super shift assay withanti-STAT1 antibody.

Cloning of a Gene Encoding 9F3 Anti-IFN-α Monoclonal Antibody

The murine anti-human IFN-α mAb 9F3 was generated, cloned and sequenced.The plasmid pEMX1 used for expression and mutagenesis of F(ab)s in E.coli has been described previously (Werther et al., J. Immunol. 157:4986-4995 [1996]). Briefly, the plasmid contains a DNA fragment encodinga consensus human κ subgroup 1 light chain (VLκl-CL) and a consensushuman subgroup III heavy chain (VHIII-CH1) and an alkaline phosphatasepromoter. The use of the consensus sequences for VL and VH has beendescribed previously (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-4289 [1992]).

Results

We have previously shown that there is a wide spectrum of IFN-α subtypesexpressed by the islets of patients with IDDM (Huang et al., Diabetes44: 658-664 [1995]). We also demonstrated that there is no obviousassociation between IDDM and the expression of either IFN-β or IFN-γ(Huang et al., [1995] supra). While the specific IFN-α subtypesexpressed as part of the SLE pathology have not been defined, as withIDDM, the association is with IFN-α and not with either of IFN-β orIFN-γ (Hooks, et al., Arthritis & Rheumatism 25: 396-400 [1982]; Kim, etal., Clin. Exp. Immunol. 70: 562-569 [1987]; Lacki, et al., J. Med. 28:99-107 [1997]; Robak, et al., Archivum Immunologiae et TherapiaeExperimentalis 46: 375-380 [1998]; Shiozawa, et al., Arthritis &Rheumatism 35: 417-422 [1992]; von Wussow, et al., RheumatologyInternational 8: 225-230 [1988]). These observations led us to proposethat a candidate antibody for therapeutic intervention in IDDM or SLEwould need to neutralize a majority of the IFN-α subtypes while leavingintact the activities of other interferons (β, γ and ω) and interleukinsthat may be required for host defense.

One of them (9F3) was able to neutralize a wide spectrum of recombinantinterferon α subtypes and was further characterized. As shown in FIG.2A, 9F3 was able to neutralize the anti-viral activity of sevenrecombinant interferons, IFN-α-2, 4, 5, 8 and 10 (FIG. 2) and IFN-α 1and 21 (Table 2 and FIG. 6). These IFN-α subtypes cover the fullspectrum of sequences as projected in a type I interferon sequencedendrogram. More importantly, the 9F3 mAb that neutralized the IFN-αsubtypes was unable to neutralize IFN-β (FIG. 2, Table 2) or IFN-γ. Thesmall increase in activity shown in FIG. 2 for IFN-β was notreproducible in other assays and appears to be the result of assayvariation.

Other mAbs that are neutralizing toward IFN-α have been developed(Tsukui et al., Microbiol. Immunol. 30: 1129-1139 [1986]; Berg, J.Interferon Res. 4: 481-491 [1984]; Meager and Berg, J. Interferon Res.6:729-736 [1986]; U.S. Pat. No. 4,902,618; and EP publication No.0,139,676 B1). However, these antibodies neutralize only a limitednumber of recombinant IFN-α subtypes and are unable to neutralize a widespectrum of IFN-α subtypes such as those produced by activatedleukocytes. In contrast, 9F3 Mab was able to neutralize at least 95% ofthe anti-viral activity in the heterogeneous collection of IFN-αsubtypes produced by activated leukocytes (FIG. 3A). Similarly, 9F3 mAbwas also able to block the anti-viral activity of an independentpreparation of lymphoblastoid IFN (NIH reference standard) as determinedin an independent experiment (FIG. 3B).

The ability of 9F3 mAb to neutralize IFN-α was also tested using analternative bioassay. The assay was based on the ability of IFN-α toactivate the binding of the signaling molecule, interferon-stimulatedgene factor 3 (ISGF3), to an oligonucleotide derived from theinterferon-stimulated response element (ISRE) in a DNA binding assayknown as electrophoretic mobility shift assay (Horvath et al., GenesDev. 9: 984-994 [1995]). The transduction of type I interferon signalsto the nucleus relies on activation of a protein complex, ISGF3,involving two signal transducers and activators of transcription (STAT)proteins, STAT1 and STAT2, and the interferon regulatory factor (IRF)protein, p48/ISGF3γ (Wathelet et al., Mol. Cell. 1: 507-518 [1998]). Thelatter is a DNA sequence recognition subunit of ISGF3 and directlyinteracts with ISRE (McKendry et al., Proc. Natl. Acad. Sci. USA 88:11455-11459 [1991]; John et al., Mol. Cell. Biol. 11: 4189-4195 [1991]).The treatment of COS cells with either IFN-α or IFN-β led to theappearance of a complex corresponding to the binding of ISGF3 to theISRE derived probe. The appearance of the IFN-α-induced but not theIFN-β-induced complex was blocked by 9F3 mAb (FIG. 4). Furthermore, 9F3mAb was able to neutralize the activity of six recombinant IFN-αsubtypes that were tested in this assay (Table 2).

TABLE 2 Inhibition of ISGF3 formation induced by type I IFNs by mAb 9F3IFN- IFN- IFN- IFN- IFN- IFN- IFN- mAb α2/1 α1 α2 α5 α8 α21 β 9F3.18.5+++ +++ +++ + +++ +++ − IgG1 − − − − − − − The extent of inhibition ofthe IFN induced complex by 9F3 is indicated where − indicates that theinduced band was not altered; + indicates that the band was partiallylost and +++ indicates that the induced band was largely abolished. mAbwas used at 10 μg/ml; IFN-α was used at 25 ng/ml

Having established that 9F3 was able to neutralize both a wide varietyof recombinant IFN-α subtypes and the mixture of IFN-α subtypes producedby activated leukocytes, we cloned and sequenced the cDNAs encoding boththe heavy and light chains of 9F3 mAb. The heavy and light chains werepurified and the N terminal amino acid sequences derived were used todesign degenerate 5′ primers corresponding to the N terminus, and the 3′primers were designed corresponding to the constant domain of mouse κlight chain and IgG2 heavy chain. The corresponding cDNAs were clonedusing conventional PCR technique and the nucleotide sequence of theinserts was determined. FIG. 5 shows sequence alignment of VL (5A) andVH (5B) domains of a murine 9F3 monoclonal antibody, a humanized version(V13) and consensus sequence of the human heavy chain subgroup III andthe human κ light chain subgroup III. In order to ensure that the cDNAsthat were cloned encoded the correct Mab reflecting the specificity andcharacteristics of 9F3 mAb, recombinant chimeric proteins were generatedthat utilized the mouse cDNA sequences shown in FIG. 5 and a human CH1domain. The resultant chimera (CH8-2) was able to fully neutralizevarious recombinant IFN-α subtypes (FIG. 6). The amino acid sequencesfor the heavy and light chains were then used to generate a humanizedantibody.

Example 2 Humanization of 9F3 Pan-IFN-α Neutralizing Monoclonal AntibodyMaterials and Methods Construction of Humanized F(ab)s

To construct the first F(ab) variant of humanized 9F3, site-directedmutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82: 488-492 [1985]) wasperformed on a deoxyuridine-containing template of pEMX1. The six CDRswere changed to the murine 9F3 sequence (FIG. 5); the residues includedin each CDR were from the sequence-based CDR definitions (Kabat et al.,(1991) supra). F-1 therefore consisted of a complete human framework(VLκ subgroup 1 and VH subgroup III) with the six complete murine CDRsequences. Plasmids for all other F(ab) variants were constructed fromthe plasmid template of F-1. Plasmids were transformed into E. colistrain XL-1 Blue (Stratagene, San Diego, Calif.) for preparation ofdouble- and single-stranded DNA using commercial kits (Qiagen, Valencia,Calif.). For each variant, DNA coding for light and heavy chains wascompletely sequenced using the dideoxynucleotide chain terminationmethod (Sequenase, U.S. Biochemical Corp., Cleveland, Ohio). Plasmidswere transformed into E. coli strain 16C9, a derivative of MM294, platedonto Luria broth plates containing 50 μg/ml carbenicillin, and a singlecolony selected for protein expression. The single colony was grown in 5ml Luria broth-100 μg/ml carbenicillin for 5-8 h at 37° C. The 5 mlculture was added to 500 ml AP5 medium containing 50 μg/ml carbenicillinand allowed to grow for 20 h in a 4 L baffled shake flask at 30° C. AP5medium consists of: 1.5 g glucose, 11.0 g Hycase SF, 0.6 g yeast extract(certified), 0.19 g MgSO₄ (anhydrous), 1.07 g NH₄C1, 3.73 g KCl, 1.2 gNaCl, 120 ml 1 M triethanolamine, pH 7.4, to 1 L water and then sterilefiltered through 0.1 μm Sealkeen filter. Cells were harvested bycentrifugation in a 1 L centrifuge bottle at 3000×g and the supernatantremoved. After freezing for 1 h, the pellet was resuspended in 25 mlcold 10 mM Tris-1 mM EDTA-20% sucrose, pH 7.5, 250 μl of 0.1 Mbenzamidine (Sigma, St. Louis, Mo.) was added to inhibit proteolysis.After gentle stirring on ice for 3 h, the sample was centrifuged at40,000×g for 15 min. The supernatant was then applied to a ProteinG-Sepharose CL-4B (Pharmacia, Uppsala, Sweden) column (0.5 ml bedvolume) equilibrated with 10 mM Tris-1 mM EDTA, pH 7.5. The column waswashed with 10 ml of 10 mM Tris-1 mM EDTA, pH 7.5, and eluted with 3 ml0.3 M glycine, pH 3.0, into 1.25 ml 1 M Tris, pH 8.0. The F(ab) was thenbuffer exchanged into PBS using a Centricon-30 (Amicon, Beverly, Mass.)and concentrated to a final volume of 0.5 ml. SDS-PAGE gels of allF(ab)s were run to ascertain purity and the molecular weight of eachvariant was verified by electrospray mass spectrometry. F(ab)concentrations were determined using quantitative amino acid analysis.

Construction of Chimeric and Humanized IgG

For generation of human IgG2 versions of chimeric and humanized 9F3, theappropriate murine or humanized VL and VH (F-13, Table 3) domains weresubcloned into separate previously described pRK vectors (Eaton et al.,Biochemistry 25: 8343-8347 [1986]) that contained DNA coding for humanIgG2 CH1-Fc or human light chain CL domain. The DNA coding for theentire light and the entire heavy chain of each variant was verified bydideoxynucleotide sequencing. The chimeric IgG consists of the entiremurine 9F3 VH domain fused to a human CH1 domain at amino acid SerH113and the entire murine 9F3 VL domain fused to a human CL domain at aminoacid LysL 107.

Heavy and light chain plasmids were co-transfected into anadenovirus-transformed human embryonic kidney cell line, 293 (Graham etal., J. Gen. Virol. 36: 59-74 [1977]), using a high efficiency procedure(Gorman et al., DNA Prot. Eng. Tech. 2: 3-10 [1990]). Media was changedto serum-free and harvested daily for up to five days. Antibodies werepurified from the pooled supernatants using Protein A-Sepharose CL-4B(Pharmacia). The eluted antibody was buffer exchanged into PBS using aCentricon-30 (Amicon), concentrated to 0.5 ml, sterile filtered using aMillex-GV (Millipore, Bedford, Mass.) and stored at 4° C. IgG2concentrations were determined using quantitative amino acid analysis.

IFN-α Binding Assay

In the ELISA, 96 well microtiter plates (Nunc) were coated by adding 50μl of 0.1 μg/ml IFN-α in PBS to each well and incubated at 4° C.overnight. The plates were then washed three times with wash buffer (PBSplus 0.05% Tween 20). The wells in microtiter plates were then blockedwith 200 μl of SuperBlock (Pierce) and incubated at room temperature for1 hour. The plates were then washed again three times with wash buffer.After washing step, 100 μl of serial dilutions of humanized mAb startingat 10 μg/ml were added to designated wells. The plates were incubated atroom temperature for 1 hour on a shaker apparatus and then washed threetimes with wash buffer. Next, 100 μA of horseradish peroxidase(HRP)-conjugated goat anti-human Fab specific (Cappel), diluted at1:1000 in assay buffer (0.5% bovine serum albumin, 0.05% Tween 20 inPBS), was added to each well. The plates were incubated at roomtemperature on a shaker apparatus and then washed three times with washbuffer, followed by addition of 100 μA of substrate (TMB,3,3′,5,5′-tetramethylbenzidine; Kirkegaard & Perry) to each well andincubated at room temperature for 10 minutes. The reaction was stoppedby adding 100 μl of stop solution (from Kirkegaard & Perry) to eachwell, and absorbance at 450 nm was read in an automated microtiter platereader.

BIAcore™ Biosensor Assay

IFN-α binding of the humanized F(ab)s, chimeric and humanized IgG2antibodies were measured using a BIACore™ biosensor (Karlsson et al.,Methods: A companion to Methods in Enzymology 6: 97-108 [1994]). TheIFN-α was immobilized on the sensor chip at 60 μg/ml in 50 mM MESbuffer, pH 6.3. Antibodies were exposed to the chip at 75 μg/ml (500 nM)in phosphate-buffered saline/1% Tween-20. The antibody on-rate (k_(on))was measured.

Computer Graphics Modes of Murine and Humanized F(ab)s

Sequences of the VL and VH domains (FIGS. 5A and B) were used toconstruct a computer graphics model of the murine 9F3 VL-VH domains(FIG. 7). This model was used to determine which framework residuesshould be incorporated into the humanized antibody. A model of thehumanized F(ab) was also constructed to verify correct selection ofmurine framework residues. Construction of models was performed asdescribed previously (Carter et al., [1992] supra; Werther et al.,[1996] supra).

Results

The consensus sequence for the human heavy chain subgroup III and thelight chain subgroup I were used as the framework for the humanizationas shown in FIG. 5 (Kabat et al., (1991), supra). This framework hasbeen successfully used in the humanization of other murine antibodies(Carter et al., Proc. Natl. Acad. Sci. USA 89: 4285-4289 [1992]; Prestaet al., J. Immunol. 151: 2623-2632 [1993]; Eigenbrot et al., Proteins18: 49-62 [1994]; Werther et al., J. Immunol. 157: 4986-4995 [1996]).All humanized variants were initially made and screened for binding asF(ab)s expressed in E. coli. Typical yields from 500 ml shake flaskswere 0.1-0.4 mg F(ab).

The complementarity determining region (CDR) residues have been definedeither based on sequence hypervariability (Kabat et al., (1991) supra)or crystal structure of F(ab)-antigen complexes (Chothia et al., Nature342: 877-883 [1989]). Although the sequence-based CDRs are larger thanthe structure-based CDRs, the two definitions are generally in agreementexcept for CDR-H1. According to the sequence-based definition, CDR-H1includes residues H31-H35, whereas the structure-based system definesresidues H26-H32 as CDR-H1 (light chain residue numbers are prefixedwith L; heavy chain residue numbers are prefixed with H). For thepresent study, CDR-H1 was defined as a combination of the two, i.e.including residues H26-H35. The other CDRs were defined using thesequence-based definition (Kabat et al., (1991) supra).

In the initial variant, F-1, the CDR residues were transferred from themurine antibody to the human framework. In addition, F(ab)s whichconsisted of the chimeric heavy chain with F-1 light chain (Ch-1) andF-1 heavy chain with chimeric light chain (Ch-2) were generated andtested for binding. F-1 bound IFN-α poorly (Table 3). Comparing thebinding affinities of Ch-1 and Ch-2 (Table 3) suggested that frameworkresidues in the F-1 VH domain needed to be altered in order to increasebinding.

TABLE 3 Humanized Anti-IFN-α Versions OD_(450 nm) at 10 μg/ml VersionTemplate Changes^(a) Mean SD N Ch-1 F-1 VL/ 1.45 0.11 3 Murine VH Ch-2Murine VL/ .024 0.04 3 F-2 VH F-1 Human FR/ 0.06 0.00 3 CDR swap F-2 F-1ArgH71Leu; 0.08 0.01 3 AsnH73Lys F-3 F-2 PheH67A1a; 0.14 0.02 3I1eH69Leu; LeuH78A1a F-4 F-3 ArgH94Ser 0.495 0.02 3 F-5 F-4 AlaH24Thr0.545 0.03 3 F-6 F-5 ValH48I1e; 0.527 0.02 2 A1aH49G1y F-7 F-5

H78Leu 0.259 0.02 2 F-8 F-5

H69I1e 0.523 0.05 3 F-9 F-5

H67Phe 0.675 0.09 3 F-10 F-9

H69I1e 0.690 0.03 3 F-11 F-10 LysH75Ser 0.642 0.06 3 F-12 F-10 AsnH76Arg0.912 0.05 3 F-13 F-12 LeuL46Val 1.050 0.16 3 TyrL49Ser F-14 F-13

H71Arg 0.472 0.06 3 F-15 F-13

H73Asn 0.868 0.32 3 ^(a)Murine residues are in bold; residue numbers areaccording to Kabat et al. (1991). Standard text indicates a change froma human framework residue to mouse. Italic text indicates a change froma mouse framework residue to human. Fab binding to IFN-α was assayed byELISA and results are provided as OD_(450 nm) at 10 μg/ml. SD, standarddeviation; n, number of experimental replicates.

Previous humanizations (Xiang et al., J. Mol. Biol. 253: 385-390 [1995];Werther et al., [1996] supra) as well as studies of F(ab)-antigencrystal structures (Chothia et al., [1989] supra; Tramontano et al., J.Mol. Biol. 215: 175-182 [1990]) have shown that residues H71 and H73 canhave a profound effect on binding, possibly by influencing theconformations of CDR-H1 and CDR-H2. Changing the human residues atpositions H71 and H73 to their murine counterparts improved binding onlyslightly (version F-2, Table 3). Further simultaneous changes atpositions H67, H69 and H78 (version F-3) followed by changes ArgH94Ser(version F-4) and AlaH24Thr (version F-5) significantly improved binding(Table 3). Since positions H67, H69 and H78 had been changedsimultaneously, each was individually altered back to the humanconsensus framework residue; versions F-7, F-8, F-9, and F-10 show thatthe human residue is preferred at position H67, position H69 does notshow any preference for the human or murine residue, and the murineresidue is preferred at position H78.

We have found during previous humanizations that residues in a frameworkloop, FR-3 (Kabat et al., (1991) supra), adjacent to CDR-H1 and CDR-H2can affect binding (Eigenbrot et al., (1994) supra). Accordingly, tworesidues in this loop were changed to their murine counterparts: LysH75to murine Ser (version F-11) and AsnH76 to murine Arg (version F-12).Only the AsnH76Arg change effected an improvement in binding (Table 3).

Inspection of the models of the murine and humanized F(ab)s suggestedthat residue L46, buried at the VL-VH interface and interacting withCDR-H3, might also play a role either in determining the conformation ofCDR-H3 and/or affecting the interactions between the VL and VH domains.Similarly, L49 position which is adjacent to CDR-L2 differs between thehuman consensus (Tyr) and the 9F3 (Ser) sequence. Therefore, LeuL46Valand TyrL49Ser residues were simultaneously substituted, which resultedin a variant (F-13) with further improvement in the binding (Table 3).Based on its best binding among all the variants generated, F-13 waschosen as the final humanized version.

A humanized recombinant anti-IFN-α monoclonal antibody (V13IgG2) wasgenerated by fusing VH and VL domains derived from F-13 to human IgG2CH₁-Fc and human CL domains respectively. The K_(ON) rates and K_(D)values of V13IgG2 were then compared with a chimeric IgG2 or murine 9F3.BIACore™ measurement of V13IgG2 and chimeric IgG2 binding to immobilizedIFN-α showed that their K_(ON) rates were similar (Table 4). Affinitymeasurement using Kinexa™ technology showed that the affinity of V13IgG2for IFNα was reduced by 2-fold compared to the parental murine 9F3antibody (Table 4).

TABLE 4 BIACore ™ and Kinexa ™ Data for Anti-IFNα AntibodiesAntibody^(a) Kd (nM)^(b) Method K_(on) (μM/sec) 0.14 BIACore ™ ChIgG23.9 BIACore ™ V13IgG2 3.3 BIACore ™ V13Fab 4.1 BIACore ™ K_(D) (pM)murine 9F3 1.5 Kinexa ™ V13Fab 3.4 Kinexa ™ ^(a)V13IgG2 is F-13 VHdomain joined to human IgG2 CH1-Fc and F-13 VL domain joined to a humanCL domain; ChIgG2 is mouse 9F3 VH domain joined to human IgG2 CH1-Fc andmouse 9F3 VL domain joined to human CL domain. ^(b)Koff/Kon.

Deposit of Material

The following materials have been deposited with the American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209,USA (ATCC):

Material ATCC Dep. No. Deposit Date 1. A hybridoma cell line PTA-2917Jan. 18, 2001 secreting 9F3 murine anti- IFN-α monoclonal antibodies(Id. Ref.: 9F3.18.5) 2. pRK-based vector for the PTA-2883 Jan. 9, 2001expression of heavy chain of chimeric CH8-2 full-length IgG (Id. Ref.:XAIFN-ChHpDR2) 3. pRK-based vector for the PTA-2880 Jan. 9, 2001expression of light chain of chimeric CH8-2 full-length IgG (Id. Ref.:XAIFN-ChLpDR1) 4. pRK-based vector for the PTA-2881 Jan. 9, 2001expression of heavy chain of humanized V13 full-length IgG₂ (Id. Ref.:VHV30-IgG2) 5. pRK-based vector for the PTA-2882 Jan. 9, 2001 expressionof light chain of humanized V13 full-length IgG₂ (Id. Ref.: VLV30-IgG)

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 U.S.C. §122 and the Commissioner's rulespursuant thereto (including 37 C.F.R. §1.14 with particular reference to8860G 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. An anti-IFN-α monoclonal antibody which binds to and neutralizes abiological activity of at least IFN-α subtypes, IFN-α1, IFN-α2, IFN-α4,IFN-α5, IFN-α8, IFN-α10, and IFN-α21.
 2. The antibody of claim 1 whichis a murine antibody.
 3. The antibody of claim 1 which is a humanizedantibody.
 4. The antibody of claim 1 which is a human antibody.
 5. Theantibody of claim 1 wherein said biological activity is antiviralactivity.
 6. The antibody of claim 5 wherein said antibody is capable ofneutralizing at least 70% of the antiviral activity of said IFN-αsubtypes.
 7. The antibody of claim 5 wherein said antibody is capable ofneutralizing at least 80% of the antiviral activity of said IFN-αsubtypes.
 8. The antibody of claim 5 wherein said antibody is capable ofneutralizing at least 90% of the antiviral activity of said IFN-αsubtypes.
 9. The antibody of claim 5 wherein said antibody is capable ofneutralizing at least 99% of the antiviral activity of said IFN-αsubtypes.
 10. The antibody of claim 1 which binds essentially the sameIFN-α epitope as murine anti-human IFN-α monoclonal antibody 9F3 or ahumanized or chimeric form thereof.
 11. The antibody of claim 1 which ismurine anti-human IFN-α monoclonal antibody 9F3 or a humanized orchimeric form thereof.
 12. The antibody of claim 11 which is humanizedanti-human IFN-α monoclonal antibody 9F3 version 13 (V13).
 13. Theantibody of claim 1 which binds essentially the same IFN-α epitope asthe anti-IFN-α antibody produced by the hybridoma cell line depositedwith ATCC on Jan. 18, 2001 and having accession No. PTA-2917.
 14. Theantibody of claim 1 which is of the IgG class.
 15. The antibody of claim14 which has an IgG₁, IgG₂, IgG₃, or IgG₄ isotype.
 16. The antibody ofclaim 1 which is an antibody fragment.
 17. The antibody of claim 16which is a Fab fragment.
 18. The antibody of claim 16 which is a F(ab′)₂fragment.
 19. The antibody of claim 16 which is a Fab′ fragment.
 20. Ananti-IFN-α antibody light chain or a fragment thereof, comprising thefollowing CDR's: (a) L1 of the formula RASQSVSTSSYSYMH (SEQ ID NO: 7);(b) L2 of the formula YASNLES (SEQ ID NO: 8); and (c) L3 of the formulaQHSWGIPRTF (SEQ ID NO: 9).
 21. The anti-IFN-α antibody light chainfragment of claim 20 which is the light chain variable domain.
 22. Ananti-IFN-α antibody heavy chain or a fragment thereof, comprising thefollowing CDR's: (a) H1 of the formula GYTFTEYIIH (SEQ ID NO: 10); (b)H2 of the formula SINPDYDITNYNQRFKG (SEQ ID NO: 11); and (c) H3 of theformula WISDFFDY (SEQ ID NO: 12).
 23. The anti-IFN-α antibody heavychain fragment of claim 22 which is the heavy chain variable domain. 24.An anti-IFN-α antibody comprising (A) at least one light chain or afragment thereof, comprising the following CDR's: (a) L1 of the formulaRASQSVSTSSYSYMH (SEQ ID NO: 7); (b) L2 of the formula YASNLES (SEQ IDNO: 8); and (c) L3 of the formula QHSWGIPRTF (SEQ ID NO: 9); and (B) atleast one heavy chain or a fragment thereof, comprising the followingCDR's: (a) H1 of the formula GYTFTEYIIH (SEQ ID NO: 10); (b) H2 of theformula SINPDYDITNYNQRFKG (SEQ ID NO: 11); and (c) H3 of the formulaWISDFFDY (SEQ ID NO: 12).
 25. The antibody of claim 24 having ahomo-tetrameric structure composed of two disulfide-bonded antibodyheavy chain-light chain pairs.
 26. The antibody of claim 24 which is alinear antibody.
 27. The antibody of claim 24 which is a murineantibody.
 28. The antibody of claim 24 which is a chimeric antibody. 29.The antibody of claim 24 which is a humanized antibody.
 30. The antibodyof claim 24 which is a human antibody.
 31. An isolated nucleic acidmolecule encoding an antibody of claim
 1. 32. An isolated nucleic acidmolecule encoding an antibody of claim
 11. 33. An isolated nucleic acidmolecule encoding an antibody of claim
 12. 34. An isolated nucleic acidmolecule encoding an antibody of claim
 24. 35. An isolated nucleic acidmolecule encoding an antibody light chain or light chain fragment ofclaim
 20. 36. An isolated nucleic acid molecule encoding an antibodyheavy chain or heavy chain fragment of claim
 22. 37. An isolated nucleicacid molecule comprising the light chain polypeptide-encoding nucleicacid sequence of the vector deposited with ATCC on Jan. 9, 2001 andhaving accession No. PTA-2882.
 38. An isolated nucleic acid moleculecomprising the heavy chain polypeptide-encoding nucleic acid sequence ofthe vector deposited with ATCC on Jan. 9, 2001 and having accession No.PTA-2881.
 39. A vector comprising a nucleic acid molecule according toany one of claims 31 to
 38. 40. A host cell transformed with a nucleicacid molecule according to any one of claims 31 to
 38. 41. A method ofproducing the antibody of any one of claims 1, 11, 12 and 24 comprisingculturing a host cell comprising a nucleic acid sequence encoding theantibody under conditions wherein the nucleic acid sequence is expressedto produce the antibody.
 42. A hybridoma cell line comprising a nucleicacid molecule according to any one of claims 31 to
 38. 43. A hybridomacell line deposited with ATCC on Jan. 18, 2001 and having accession No.PTA-2917.
 44. An antibody produced by the hybridoma cell line of claim42.
 45. A pharmaceutical composition comprising an effective amount ofthe antibody of claim 1 in admixture with a pharmaceutically acceptablecarrier.
 46. A pharmaceutical composition comprising an effective amountof the antibody of claim 11 in admixture with a pharmaceuticallyacceptable carrier.
 47. A pharmaceutical composition comprising aneffective amount of the antibody of claim 12 in admixture with apharmaceutically acceptable carrier.
 48. A pharmaceutical compositioncomprising an effective amount of the antibody of claim 24 in admixturewith a pharmaceutically acceptable carrier.
 49. A method for diagnosinga condition associated with the expression of IFN-α in a cell,comprising contacting said cell with an anti-IFN-α antibody of claim 1,and detecting the presence of IFN-α.
 50. A method for the treatment of adisease or condition associated with the expression of IFN-α in apatient, comprising administering to said patient an effective amount ofan anti-IFN-α antibody of claim
 1. 51. The method of claim 50 whereinsaid patient is a mammalian patient.
 52. The method of claim 51 whereinsaid patient is human.
 53. The method of claim 52 wherein said diseaseis an autoimmune disease.
 54. The method of claim 53 wherein saiddisease is selected from the group consisting of insulin-dependentdiabetes mellitus (IDDM); systemic lupus erythematosus (SLE); andautoimmune thyroiditis.